U.S. patent application number 14/414568 was filed with the patent office on 2015-07-30 for liposomal compositions of epoxyketone-based proteasome inhibitors.
The applicant listed for this patent is ONYX THERAPEUTICS, INC.. Invention is credited to Elena T. Chan, Katherine A. Chu, Ying Fang, Mouhannad Jamaa, Jing Jiang, Jeffrey Joseph Jones, Christopher Justin Kirk, Tony Muchamuel, Zhengping Wang.
Application Number | 20150209282 14/414568 |
Document ID | / |
Family ID | 49949227 |
Filed Date | 2015-07-30 |
United States Patent
Application |
20150209282 |
Kind Code |
A1 |
Chu; Katherine A. ; et
al. |
July 30, 2015 |
LIPOSOMAL COMPOSITIONS OF EPOXYKETONE-BASED PROTEASOME
INHIBITORS
Abstract
Liposomal compositions comprising peptide epoxyketone compounds
are described, as well as methods of making and using such
liposomal compositions. These liposomal compositions enhance the
therapeutic window of peptide epoxyketone compounds by improving in
vivo half-life relative to non-liposomal compositions comprising
peptide epoxyketone compounds, providing desirable pharmacodynamic
profiles, and providing anti-tumor activity in a human tumor
xenograft model, greater than or equal to non-liposomal
compositions comprising peptide epoxyketone compounds. Further,
experiments performed in support of the present invention
demonstrated improved tolerability of liposomal compositions
comprising peptide epoxyketone compounds.
Inventors: |
Chu; Katherine A.; (San
Francisco, CA) ; Chan; Elena T.; (San Mateo, CA)
; Fang; Ying; (Saratoga, CA) ; Jamaa;
Mouhannad; (Foster City, CA) ; Kirk; Christopher
Justin; (Milbrae, CA) ; Muchamuel; Tony;
(Boulder Creek, CA) ; Wang; Zhengping; (San Mateo,
CA) ; Jiang; Jing; (San Jose, CA) ; Jones;
Jeffrey Joseph; (Redwood City, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ONYX THERAPEUTICS, INC. |
South San Francisco |
CA |
US |
|
|
Family ID: |
49949227 |
Appl. No.: |
14/414568 |
Filed: |
July 17, 2013 |
PCT Filed: |
July 17, 2013 |
PCT NO: |
PCT/US13/50872 |
371 Date: |
January 13, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61794603 |
Mar 15, 2013 |
|
|
|
61673017 |
Jul 18, 2012 |
|
|
|
Current U.S.
Class: |
424/450 ;
514/20.9; 514/21.9 |
Current CPC
Class: |
A61P 25/28 20180101;
A61P 43/00 20180101; A61P 31/12 20180101; A61P 35/02 20180101; A61P
25/16 20180101; A61P 31/04 20180101; A61P 9/10 20180101; A61P 25/00
20180101; A61P 3/12 20180101; A61P 27/02 20180101; A61P 3/00
20180101; A61K 9/1277 20130101; A61K 45/06 20130101; A61P 25/14
20180101; A61P 39/02 20180101; A61P 31/10 20180101; A61P 21/00
20180101; A61K 47/6951 20170801; A61K 9/127 20130101; A61K 9/1271
20130101; A61P 31/18 20180101; A61P 19/08 20180101; A61P 35/00
20180101; A61P 37/06 20180101; A61P 11/00 20180101; A61P 29/00
20180101; A61K 47/22 20130101; A61P 33/14 20180101; A61P 17/14
20180101; A61K 38/07 20130101 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 47/48 20060101 A61K047/48; A61K 45/06 20060101
A61K045/06; A61K 38/07 20060101 A61K038/07 |
Claims
1. A pharmaceutical liposomal composition comprising: an aqueous
solution comprising liposomes; and the liposomes comprising (i)
between about 0.5 wt. % and about 50 wt. % of a peptide epoxyketone
compound, wherein the peptide epoxyketone compound is entrapped in
the liposomes; (ii) between about 99.5 wt. % and about 50 wt. %
total lipids, wherein the total lipids comprise a phospholipid
selected from the group consisting of
L-.alpha.-phosphatidylcholine;
1,2-distearoyl-sn-glycero-3-phosphocholine;
1,2-dipalmitoyl-sn-glycero-3-phosphocholine;
1,2-Distearoyl-sn-glycero-3-phospho-rac-(1-glycerol);
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; sphingomyelin;
1,2-distearoyl-sn-glycero-3-phosphoethanolamine; and combinations
thereof; wherein the liposomes have an average size of between
about 0.05 microns and about 0.5 microns.
2. The pharmaceutical liposomal composition of claim 1, wherein the
phospholipid comprises L-.alpha.-phosphatidylcholine.
3. The pharmaceutical liposomal composition of claim 1, wherein the
phospholipid comprises sphingomyelin.
4. The pharmaceutical liposomal composition of claim 1, wherein the
phospholipid comprises
1,2-distearoyl-sn-glycero-3-phosphocholine.
5. The pharmaceutical liposomal composition of claim 1, wherein the
phospholipid comprises
1,2-dipalmitoyl-sn-glycero-3-phosphocholine.
6. The pharmaceutical liposomal composition of claim 1, wherein the
phospholipid comprises
1,2-distearoyl-sn-glycero-3-phosphoethanolamine.
7. The pharmaceutical liposomal composition of claim 1, wherein the
phospholipid comprises
1,2-Distearoyl-sn-glycero-3-phospho-rac-(1-glycerol).
8. The pharmaceutical liposomal composition of claim 1, wherein the
phospholipid comprises
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.
9. The pharmaceutical liposomal composition of any preceding claim,
wherein the total lipids comprise between about 20 wt. % and about
100 wt. % of the phospholipid.
10. The pharmaceutical liposomal composition of claim 9, wherein
the total lipids comprise between about 30 wt. % and about 90 wt. %
of the phospholipid.
11. The pharmaceutical liposomal composition of claim 9, wherein
the total lipids comprise between about 50 wt. % and about 75 wt. %
of the phospholipid.
12. The pharmaceutical liposomal composition of any of claims 1-8,
wherein the total lipids further comprise a hydrophilic
polymer-derivatized lipid.
13. The pharmaceutical liposomal composition of claim 12, wherein
the total lipids comprise between about 0.1 wt. % and about 30 wt.
% of the hydrophilic polymer-derivatized lipid.
14. The pharmaceutical liposomal composition of claim 12, wherein
the total lipids comprise between about 5 wt. % and about 25 wt. %
of the hydrophilic polymer-derivatized lipid.
15. The pharmaceutical liposomal composition of claim 12, wherein
the total lipids comprise between about 8 wt. % and about 20 wt. %
of the hydrophilic polymer-derivatized lipid.
16. The pharmaceutical liposomal composition of claim 12,
comprising between about 90 wt. % of the phospholipid and about 75
wt. % of the phospholipid, and between about 10 wt. % of the
hydrophilic polymer-derivatized lipid and about 25 wt. % of the
hydrophilic polymer-derivatized lipid.
17. The pharmaceutical liposomal composition of any of claims
12-16, wherein the hydrophilic polymer-derivatized lipid comprises
a hydrophilic polymer and a lipid, and the hydrophilic polymer is a
polyethylene glycol.
18. The pharmaceutical liposomal composition of claim 17, wherein
the lipid of the hydrophilic polymer-derivatized lipid is
cholesterol or a phospholipid.
19. The pharmaceutical liposomal composition of claim 17, wherein
the lipid of the hydrophilic polymer-derivatized lipid is a
phospholipid and the hydrophilic polymer-derivatized lipid is
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000].
20. The pharmaceutical liposomal composition of any of claims 1-8,
wherein the total lipids further comprise a cholesterol or
cholesterol derivative.
21. The pharmaceutical liposomal composition of claim 20, wherein
the total lipids comprise between about 10 wt. % and about 50 wt. %
of the cholesterol or cholesterol derivative.
22. The pharmaceutical liposomal composition of claim 20, wherein
the total lipids comprise between about 15 wt. % and about 40 wt. %
of the cholesterol or cholesterol derivative.
23. The pharmaceutical liposomal composition of claim 20, wherein
the total lipids comprise between about 15 wt. % and about 30 wt. %
of the cholesterol or cholesterol derivative.
24. The pharmaceutical liposomal composition of claim 20, wherein
the total lipids comprise between about 90 wt. % and about 50 wt. %
of the phospholipid, and between about 10 wt. % and about 50 wt. %
of the cholesterol or derivative.
25. The pharmaceutical liposomal composition of any of claims
20-24, wherein the cholesterol or cholesterol derivative is
cholesterol.
26. The pharmaceutical liposomal composition of claim 20, wherein
the total lipids further comprise a hydrophilic polymer-derivatized
lipid.
27. The pharmaceutical liposomal composition of claim 26, wherein
the total lipids comprise between about 83.3 wt. % of the
phospholipid and about 57 wt. % of the phospholipid, between about
8.33 wt. % of the hydrophilic polymer-derivatized lipid and about
14 wt. % of the hydrophilic polymer-derivatized lipid, and between
about 8.33 wt. % of the cholesterol or cholesterol derivative and
about 29 wt. % of the cholesterol or cholesterol derivative.
28. The pharmaceutical liposomal composition of claim 27, wherein
the cholesterol or cholesterol derivative is cholesterol.
29. The pharmaceutical liposomal composition of any of claims
27-28, wherein the hydrophilic polymer-derivatized lipid comprises
a hydrophilic polymer and a lipid, and the hydrophilic polymer is a
polyethylene glycol.
30. The pharmaceutical liposomal composition of claim 29, wherein
the lipid of the hydrophilic polymer-derivatized lipid is a
phospholipid.
31. The pharmaceutical liposomal composition of claim 30, wherein
the hydrophilic polymer-derivatized lipid is
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000].
32. The pharmaceutical liposomal composition of any preceding
claim, comprising between about 0.5 wt. % and about 35 wt. % of the
peptide epoxyketone compound, and between about 99.5 wt. % and
about 65 wt. % of the total lipids.
33. The pharmaceutical liposomal composition of any of claims 1-32,
comprising between about 1 wt. % and about 30 wt. % of the peptide
epoxyketone compound, and between about 99 wt. % and about 70 wt. %
of the total lipids.
34. The pharmaceutical liposomal composition of any of claims 1-32,
comprising between about 1 wt. % and about 25 wt. % of the peptide
epoxyketone compound, and between about 99 wt. % and about 75 wt. %
of the total lipids.
35. The pharmaceutical liposomal composition of any preceding
claim, wherein the average size of the liposomes is between about
0.05 microns and about 0.2 microns.
36. The pharmaceutical liposomal composition of claim 35, wherein
the average size of the liposomes is selected from the group
consisting of: between about 0.05 microns and about 0.15 microns;
and between about 0.05 microns and about 0.10 microns.
37. The pharmaceutical liposomal composition of any preceding
claim, wherein the aqueous solution further comprises a pH
adjusting agent.
38. The pharmaceutical liposomal composition of any preceding
claim, wherein the aqueous solution further comprises an agent to
maintain isotonicity.
39. The pharmaceutical liposomal composition of any preceding
claim, wherein the liposomes comprises the peptide epoxyketone
compound and a solubilizing agent in an internal aqueous core of
the liposomes.
40. The pharmaceutical liposomal composition of claim 39, wherein
the solubilizing agent is a cyclodextrin, and the liposomes of the
liposomal composition comprise the peptide epoxyketone compound
complexed with the cyclodextrin in the internal aqueous core of the
liposomes.
41. The pharmaceutical liposomal composition of claim 40, wherein
the cyclodextrin is a sulfobutylether-betacyclodextrin or a
hydroxypropyl-betacyclodextrin.
42. The pharmaceutical liposomal composition of claim 41, wherein
the cyclodextrin is a sulfobutylether-betacyclodextrin.
43. The pharmaceutical liposomal composition of claim 41, wherein
the cyclodextrin is a hydroxypropyl-betacyclodextrin.
44. The pharmaceutical liposomal composition of any preceding
claim, wherein the aqueous solution of liposomal composition has a
pH between about pH 3.0 and about pH 7.0.
45. The pharmaceutical liposomal composition of claim 44, wherein
the pH is a human physiological pH.
46. The pharmaceutical liposomal composition of any preceding
claim, wherein the peptide epoxyketone compound is selected from
the group consisting of compound II, compound III, compound IV, and
compound V.
47. The pharmaceutical liposomal composition of any preceding
claim, wherein the peptide epoxyketone compound is carfilzomib
(compound V).
48. A dry pharmaceutical composition formed by drying the
pharmaceutical liposomal composition of any preceding claim.
49. A dry pharmaceutical composition comprising: between about 0.5
wt. % and about 50 wt. % of a peptide epoxyketone compound; and
(ii) between about 99.5 wt. % and about 50 wt. % total lipids,
wherein the total lipids comprise a phospholipid selected from the
group consisting of L-.alpha.-phosphatidylcholine;
1,2-distearoyl-sn-glycero-3-phosphocholine;
1,2-dipalmitoyl-sn-glycero-3-phosphocholine;
1,2-Distearoyl-sn-glycero-3-phospho-rac-(1-glycerol);
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; sphingomyelin;
1,2-distearoyl-sn-glycero-3-phosphoethanolamine; and combinations
thereof.
50. The dry pharmaceutical composition of claim 49, wherein the
total lipids comprise between about 20 wt. % and about 100 wt. % of
the phospholipid.
51. The dry pharmaceutical composition of claim 50, wherein the
total lipids comprise between about 30 wt. % and about 90 wt. % of
the phospholipid.
52. The dry pharmaceutical composition of claim 50, wherein the
total lipids comprise between about 50 wt. % and about 75 wt. % of
the phospholipid.
53. The dry pharmaceutical composition of any of claim 49, wherein
the total lipids further comprise a hydrophilic polymer-derivatized
lipid.
54. The dry pharmaceutical composition of claim 53, wherein the
total lipids comprise between about 0.1 wt. % and about 30 wt. % of
the hydrophilic polymer-derivatized lipid.
55. The dry pharmaceutical composition of claim 53, wherein the
total lipids comprise between about 5 wt. % and about 25 wt. % of
the hydrophilic polymer-derivatized lipid.
56. The dry pharmaceutical composition of claim 53, wherein the
total lipids comprise between about 8 wt. % and about 20 wt. % of
the hydrophilic polymer-derivatized lipid.
57. The dry pharmaceutical composition of claim 53, comprising
between about 90 wt. % of the phospholipid and about 75 wt. % of
the phospholipid, and between about 10 wt. % of the hydrophilic
polymer-derivatized lipid and about 25 wt. % of the hydrophilic
polymer-derivatized lipid.
58. The dry pharmaceutical composition of any of claims 53-57,
wherein the hydrophilic polymer-derivatized lipid comprises a
hydrophilic polymer and a lipid, and the hydrophilic polymer is a
polyethylene glycol.
59. The dry pharmaceutical composition of claim 58, wherein the
lipid of the hydrophilic polymer-derivatized lipid is cholesterol
or a phospholipid.
60. The dry pharmaceutical composition of claim 58, wherein the
lipid of the hydrophilic polymer-derivatized lipid is a
phospholipid and the hydrophilic polymer-derivatized lipid is
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000].
61. The dry pharmaceutical composition of claim 49, wherein the
total lipids further comprise a cholesterol or cholesterol
derivative.
62. The dry pharmaceutical composition of claim 61, wherein the
total lipids comprise between about 10 wt. % and about 50 wt. % of
the cholesterol or cholesterol derivative.
63. The dry pharmaceutical composition of claim 61, wherein the
total lipids comprise between about 15 wt. % and about 40 wt. % of
the cholesterol or cholesterol derivative.
64. The dry pharmaceutical composition of claim 61, wherein the
total lipids comprise between about 15 wt. % and about 30 wt. % of
the cholesterol or cholesterol derivative.
65. The dry pharmaceutical composition of claim 61, wherein the
total lipids comprise between about 90 wt. % and about 50 wt. % of
the phospholipid, and between about 10 wt. % and about 50 wt. % of
the cholesterol or derivative.
66. The dry pharmaceutical composition of any of claims 61-65,
wherein the cholesterol or cholesterol derivative is
cholesterol.
67. The dry pharmaceutical composition of claim 61, wherein the
total lipids further comprise a hydrophilic polymer-derivatized
lipid.
68. The dry pharmaceutical composition of claim 67, wherein the
total lipids comprise between about 83.3 wt. % of the phospholipid
and about 57 wt. % of the phospholipid, between about 8.33 wt. % of
the hydrophilic polymer-derivatized lipid and about 14 wt. % of the
hydrophilic polymer-derivatized lipid, and between about 8.33 wt. %
of the cholesterol or cholesterol derivative and about 29 wt. % of
the cholesterol or cholesterol derivative.
69. The dry pharmaceutical composition of claim 68, wherein the
cholesterol or cholesterol derivative is cholesterol.
70. The dry pharmaceutical composition of any of claims 67-69,
wherein the hydrophilic polymer-derivatized lipid comprises a
hydrophilic polymer and a lipid, and the hydrophilic polymer is a
polyethylene glycol.
71. The dry pharmaceutical composition of claim 70, wherein the
lipid of the hydrophilic polymer-derivatized lipid is a
phospholipid.
72. The dry pharmaceutical composition of claim 71, wherein the
hydrophilic polymer-derivatized lipid is
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000].
73. The dry pharmaceutical composition of any of claims 49-72,
comprising between about 0.5 wt. % and about 35 wt. % of the
peptide epoxyketone compound, and between about 99.5 wt. % and
about 65 wt. % of the total lipids.
74. The dry pharmaceutical composition of any of claims 49-72,
comprising between about 1 wt. % and about 30 wt. % of the peptide
epoxyketone compound, and between about 99 wt. % and about 70 wt. %
of the total lipids.
75. The dry pharmaceutical composition of any of claims 49-72,
comprising between about 1 wt. % and about 25 wt. % of the peptide
epoxyketone compound, and between about 99 wt. % and about 75 wt. %
of the total lipids.
76. The dry pharmaceutical composition of any preceding claim,
further comprising a pH adjusting agent.
77. The dry pharmaceutical composition of any preceding claim,
further comprising an agent to maintain isotonicity.
78. The dry pharmaceutical composition of any preceding claim,
further comprising a cyclodextrin.
79. The dry pharmaceutical composition of claim 78, wherein the
cyclodextrin is a sulfobutylether-betacyclodextrin or a
hydroxypropyl-betacyclodextrin.
80. The dry pharmaceutical composition of claim 79, wherein the
cyclodextrin is a sulfobutylether-betacyclodextrin.
81. The dry pharmaceutical composition of claim 79, wherein the
cyclodextrin is a hydroxypropyl-betacyclodextrin.
82. The dry pharmaceutical composition of any preceding claim,
wherein the peptide epoxyketone compound is selected from the group
consisting of compound II, compound III, compound IV, and compound
V.
83. The dry pharmaceutical composition of any preceding claim,
wherein the peptide epoxyketone compound is carfilzomib (compound
V).
84. The dry pharmaceutical composition of any preceding claim,
further comprising additional excipients.
85. The dry pharmaceutical composition of claim 84, wherein the
additional excipients comprise one or more excipient selected from
the group consisting of a cryoprotectant agent, a sugar, a glass
transition modifying agent, and a combination thereof.
86. A method of making a pharmaceutical liposomal composition
comprising: reconstituting a dry pharmaceutical composition of any
of claims 49-85 with an aqueous solution to form liposomes, the
pharmaceutical liposomal composition comprising the aqueous
solution comprising the liposomes.
87. A pharmaceutical liposomal composition made by the method of
claim 86.
88. The pharmaceutical liposomal composition of claim 87, wherein
the liposomes have an average size of between about 0.05 microns
and about 0.5 microns.
89. The pharmaceutical liposomal composition of claim 88, wherein
the average size of the liposomes is between about 0.05 microns and
about 0.2 microns.
90. The pharmaceutical liposomal composition of claim 89, wherein
the average size of the liposomes is selected from the group
consisting of: between about 0.05 microns and about 0.15 microns;
and between about 0.05 microns and about 0.10 microns.
91. A method of making a pharmaceutical liposomal composition
comprising: preparing a dried film comprising total lipids, wherein
the total lipids comprise a phospholipid selected from the group
consisting of L-.alpha.-phosphatidylcholine;
1,2-distearoyl-sn-glycero-3-phosphocholine;
1,2-dipalmitoyl-sn-glycero-3-phosphocholine;
1,2-Distearoyl-sn-glycero-3-phospho-rac-(1-glycerol);
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; sphingomyelin;
1,2-distearoyl-sn-glycero-3-phosphoethanolamine; and a combination
thereof; and rehydrating the dried film with an aqueous solution
comprising a peptide epoxyketone compound and a solubilizing agent
to form the liposomal composition comprising liposomes dispersed in
the aqueous solution; wherein the liposomes comprise (i) between
about 0.5 wt. % and about 50 wt. % of the peptide epoxyketone
compound entrapped in the liposomes, and (ii) between about 99.5
wt. % and about 50 wt. % of the total lipids.
92. The method of claim 91, wherein the total lipids comprise
between about 20 wt. % and about 100 wt. % of the phospholipid.
93. The method of claim 92, wherein the total lipids comprise
between about 30 wt. % and about 90 wt. % of the phospholipid.
94. The method of claim 93, wherein the total lipids comprise
between about 50 wt. % and about 75 wt. % of the phospholipid.
95. The method of claim 91, wherein the total lipids further
comprise a hydrophilic polymer-derivatized lipid.
96. The method of claim 95, wherein the total lipids comprise
between about 0.1 wt. % and about 30 wt. % of the hydrophilic
polymer-derivatized lipid.
97. The method of claim 95, wherein the total lipids comprise
between about 5 wt. % and about 25 wt. % of the hydrophilic
polymer-derivatized lipid.
98. The method of claim 95, wherein the total lipids comprise
between about 8 wt. % and about 20 wt. % of the hydrophilic
polymer-derivatized lipid.
99. The method of claim 95, wherein the total lipids comprise
between about 90 wt. % of the phospholipid and about 75 wt. % of
the phospholipid, and between about 10 wt. % of the hydrophilic
polymer-derivatized lipid and about 25 wt. % of the hydrophilic
polymer-derivatized lipid.
100. The method of any of claims 95-99, wherein the hydrophilic
polymer-derivatized lipid comprises a hydrophilic polymer and a
lipid, and the hydrophilic polymer is a polyethylene glycol.
101. The method of claim 100, wherein the lipid of the hydrophilic
polymer-derivatized lipid is cholesterol or a phospholipid.
102. The method of claim 100, wherein the hydrophilic
polymer-derivatized lipid is
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polye-
thylene glycol)-2000].
103. The method of claim 91, wherein the total lipids further
comprise a cholesterol or cholesterol derivative.
104. The method of claim 103, wherein the total lipids comprise
between about 10 wt. % and about 50 wt. % of the cholesterol or
cholesterol derivative.
105. The method of claim 103, wherein the total lipids comprise
between about 15 wt. % and about 40 wt. % of the cholesterol or
cholesterol derivative.
106. The method of claim 103, wherein the total lipids comprise
between about 15 wt. % and about 30 wt. % of the cholesterol or
cholesterol derivative.
107. The method of claim 103, wherein the total lipids comprise
between about 90 wt. % and about 50 wt. % of the phospholipid, and
between about 10 wt. % and about 50 wt. % of the cholesterol or
derivative.
108. The method of any of claims 103-107, wherein the cholesterol
or cholesterol derivative is cholesterol.
109. The method of claim 103, wherein the total lipids further
comprise a hydrophilic polymer-derivatized lipid.
110. The method of claim 109, wherein the total lipids comprise
between about 83.3 wt. % of the phospholipid and about 57 wt. % of
the phospholipid, between about 8.33 wt. % of the hydrophilic
polymer-derivatized lipid and about 14 wt. % of the hydrophilic
polymer-derivatized lipid, and between about 8.33 wt. % of the
cholesterol or cholesterol derivative and about 29 wt. % of the
cholesterol or cholesterol derivative.
111. The method of claim 110, wherein the cholesterol or
cholesterol derivative is cholesterol.
112. The method of any of claims 109-111, wherein the hydrophilic
polymer-derivatized lipid comprises a hydrophilic polymer and a
lipid, and the hydrophilic polymer is a polyethylene glycol.
113. The method of claim 112, wherein the lipid of the hydrophilic
polymer-derivatized lipid is a phospholipid.
114. The method of claim 112, wherein the hydrophilic
polymer-derivatized lipid is
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polye-
thylene glycol)-2000].
115. The method of any of claims 91-114, wherein the liposomes
comprise (i) between about 0.5 wt. % and about 35 wt. % of the
peptide epoxyketone compound entrapped in the liposomes, and (ii)
between about 99.5 wt. % and about 65 wt. % of the total
lipids.
116. The method of any of claims 91-114, wherein the liposomes
comprise (i) between about 1 wt. % and about 30 wt. % of the
peptide epoxyketone compound entrapped in the liposomes, and (ii)
between about 99 wt. % and about 70 wt. % of the total lipids.
117. The method of any of claims 91-114, wherein the liposomes
comprise (i) between about 1 wt. % and about 25 wt. % of the
peptide epoxyketone compound entrapped in the liposomes, and (ii)
between about 99 wt. % and about 75 wt. % of the total lipids.
118. The method of any of claims 91-117, wherein the solubilizing
agent is selected from the group consisting of a compound, a pH
adjusting agent, a cosolvent, and a combination thereof.
119. The method of claim 118, wherein the solubilizing agent
comprises a compound, the compound is a cyclodextrin, and the
liposomes of the liposomal composition comprise the peptide
epoxyketone compound complexed with the cyclodextrin in an internal
aqueous core of the liposomes.
120. The method of claim 119, wherein the cyclodextrin is a
sulfobutylether-betacyclodextrin or a
hydroxypropyl-betacyclodextrin.
121. The method of any of claims 91-120, wherein the solubilizing
agent comprises a pH adjusting agent and the aqueous solution has a
pH of between about pH 0.5 and about pH 3.
122. The method of claim 121, wherein the pH of the aqueous
solution is between about pH 0.5 and about pH 2.
123. The method of claim 122, wherein the pH of the aqueous
solution is between about pH 1 and about pH 2.
124. The method of any of claims 91-123, wherein the solubilizing
agent comprises a cosolvent.
125. The method of any of claims 91-124, further comprising
dialysis, desalting, buffer exchange, or gel filtration.
126. The method of any of claims 91-125, further comprising sizing
the liposomes to have an average size of between about 0.05 microns
and about 0.5 microns.
127. The method of claim 126, comprising sizing the liposomes to
have an average size of between about 0.05 microns and about 0.2
microns.
128. The method of claim 127, comprising sizing the liposomes to
have an average size selected from the group consisting of: between
about 0.05 microns and about 0.15 microns; and between about 0.05
microns and about 0.10 microns.
129. The method of any of claims 91-128, wherein the liposomal
composition comprises encapsulated aqueous solution and
non-encapsulated aqueous solution.
130. The method of claim 129, further comprising, after rehydrating
the dried film to form the liposomal composition, removing the
peptide epoxyketone compound from the non-encapsulated aqueous
solution.
131. The method of claim 130, wherein dialysis,
ultracentrifugation, or gel filtration is used for removing the
peptide epoxyketone compound from the non-encapsulated aqueous
solution.
132. The method of any of claims 91-131, further comprising, after
rehydrating the dried film to form the liposomal composition,
adjusting the aqueous solution to a pH of between about pH 3.0 and
about pH 7.0.
133. The method of any of claims 91-131, further comprising, after
rehydrating the dried film to form the liposomal composition,
adjusting the aqueous solution to a human physiological pH.
134. The method of any of claims 91-133, further comprising, after
rehydrating the dried film to form the liposomal composition,
adding an agent to maintain isotonicity to the aqueous
solution.
135. The method of any of claims 91-134, wherein the peptide
epoxyketone compound is selected from the group consisting of
compound II, compound III, compound IV, and compound V.
136. The method of any of claims 91-134, wherein the peptide
epoxyketone compound is carfilzomib (compound V).
137. A pharmaceutical liposomal composition made by the method of
any of claims 91-136, the liposomal composition comprising
liposomes dispersed in the aqueous solution wherein the liposomes
comprise the peptide epoxyketone compound entrapped in the
liposomes.
138. A dry pharmaceutical composition formed by drying the
pharmaceutical liposomal composition of claim 137.
139. A method of making a pharmaceutical liposomal composition
comprising: preparing a lipid solution comprising a solvent and
total lipids, wherein the total lipids comprise a phospholipid
selected from the group consisting of
L-.alpha.-phosphatidylcholine;
1,2-distearoyl-sn-glycero-3-phosphocholine;
1,2-dipalmitoyl-sn-glycero-3-phosphocholine;
1,2-Distearoyl-sn-glycero-3-phospho-rac-(1-glycerol);
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; sphingomyelin;
1,2-distearoyl-sn-glycero-3-phosphoethanolamine; and a combination
thereof; and injecting the lipid solution into an aqueous solution
comprising a peptide epoxyketone compound and a solubilizing agent
to form the liposomal composition comprising liposomes dispersed in
the aqueous solution; wherein the liposomes comprise (i) between
about 0.5 wt. % and about 50 wt. % of the peptide epoxyketone
compound entrapped in the liposomes, and (ii) between about 99.5
wt. % and about 50 wt. % of the total lipids.
140. The method of claim 139, wherein the total lipids comprise
between about 20 wt. % and about 100 wt. % of the phospholipid.
141. The method of claim 140, wherein the total lipids comprise
between about 30 wt. % and about 90 wt. % of the phospholipid.
142. The method of claim 141, wherein the total lipids comprise
between about 50 wt. % and about 75 wt. % of the phospholipid.
143. The method of claim 139, wherein the total lipids further
comprise a hydrophilic polymer-derivatized lipid.
144. The method of claim 143, wherein the total lipids comprise
between about 0.1 wt. % and about 30 wt. % of the hydrophilic
polymer-derivatized lipid.
145. The method of claim 143, wherein the total lipids comprise
between about 5 wt. % and about 25 wt. % of the hydrophilic
polymer-derivatized lipid.
146. The method of claim 143, wherein the total lipids comprise
between about 8 wt. % and about 20 wt. % of the hydrophilic
polymer-derivatized lipid.
147. The method of claim 143, wherein the total lipids comprise
between about 90 wt. % of the phospholipid and about 75 wt. % of
the phospholipid, and between about 10 wt. % of the hydrophilic
polymer-derivatized lipid and about 25 wt. % of the hydrophilic
polymer-derivatized lipid.
148. The method of any of claims 143-147, wherein the hydrophilic
polymer-derivatized lipid comprises a hydrophilic polymer and a
lipid, and the hydrophilic polymer is a polyethylene glycol.
149. The method of claim 148, wherein the lipid of the hydrophilic
polymer-derivatized lipid is cholesterol or a phospholipid.
150. The method of claim 148, wherein the hydrophilic
polymer-derivatized lipid is
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polye-
thylene glycol)-2000].
151. The method of claim 139, wherein the total lipids further
comprise a cholesterol or cholesterol derivative.
152. The method of claim 151, wherein the total lipids comprise
between about 10 wt. % and about 50 wt. % of the cholesterol or
cholesterol derivative.
153. The method of claim 151, wherein the total lipids comprise
between about 15 wt. % and about 40 wt. % of the cholesterol or
cholesterol derivative.
154. The method of claim 151, wherein the total lipids comprise
between about 15 wt. % and about 30 wt. % of the cholesterol or
cholesterol derivative.
155. The method of claim 151, wherein the total lipids comprise
between about 90 wt. % and about 50 wt. % of the phospholipid, and
between about 10 wt. % and about 50 wt. % of the cholesterol or
derivative.
156. The method of any of claims 151-155, wherein the cholesterol
or cholesterol derivative is cholesterol.
157. The method of claim 151, wherein the total lipids further
comprise a hydrophilic polymer-derivatized lipid.
158. The method of claim 157, wherein the total lipids comprise
between about 83.3 wt. % of the phospholipid and about 57 wt. % of
the phospholipid, between about 8.33 wt. % of the hydrophilic
polymer-derivatized lipid and about 14 wt. % of the hydrophilic
polymer-derivatized lipid, and between about 8.33 wt. % of the
cholesterol or cholesterol derivative and about 29 wt. % of the
cholesterol or cholesterol derivative.
159. The method of claim 158, wherein the cholesterol or
cholesterol derivative is cholesterol.
160. The method of any of claims 157-159, wherein the hydrophilic
polymer-derivatized lipid comprises a hydrophilic polymer and a
lipid, and the hydrophilic polymer is a polyethylene glycol.
161. The method of claim 160, wherein the lipid of the hydrophilic
polymer-derivatized lipid is a phospholipid.
162. The method of claim 161, wherein the hydrophilic
polymer-derivatized lipid is
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polye-
thylene glycol)-2000].
163. The method of any of claims 139-162, wherein the liposomes
comprise (i) between about 0.5 wt. % and about 35 wt. % of the
peptide epoxyketone compound entrapped in the liposomes, and (ii)
between about 99.5 wt. % and about 65 wt. % of the total
lipids.
164. The method of any of claims 139-162, wherein the liposomes
comprise (i) between about 1 wt. % and about 30 wt. % of the
peptide epoxyketone compound entrapped in the liposomes, and (ii)
between about 99 wt. % and about 70 wt. % of the total lipids.
165. The method of any of claims 139-162, wherein the liposomes
comprise (i) between about 1 wt. % and about 25 wt. % of the
peptide epoxyketone compound entrapped in the liposomes, and (ii)
between about 99 wt. % and about 75 wt. % of the total lipids.
166. The method of any of claims 139-165, wherein the solvent is an
organic solvent.
167. The method of claim 166, wherein the solvent is ethanol.
168. The method of any of claims 139-167, wherein the solubilizing
agent is selected from the group consisting of a compound, a pH
adjusting agent, a cosolvent, and a combination thereof.
169. The method of claim 168, wherein the solubilizing agent
comprises a compound, the compound is a cyclodextrin, and the
liposomes of the liposomal composition comprise the peptide
epoxyketone compound complexed with the cyclodextrin in an internal
aqueous core of the liposomes.
170. The method of claim 169, wherein the cyclodextrin is a
sulfobutylether-betacyclodextrin or a
hydroxypropyl-betacyclodextrin.
171. The method of any of claims 139-170, wherein the solubilizing
agent comprises a pH adjusting agent and the aqueous solution has a
pH of between about pH 0.5 and about pH 3.
172. The method of claim 171, wherein the pH of the aqueous
solution is between about pH 0.5 and about pH 2.
173. The method of claim 172, wherein the pH of the aqueous
solution is between about pH 1 and about pH 2.
174. The method of any of claims 139-173, wherein the solubilizing
agent comprises a cosolvent.
175. The method of any of claims 139-174, further comprising
dialysis, desalting, buffer exchange, or gel filtration.
176. The method of any of claims 139-175, further comprising sizing
the liposomes to have an average size of between about 0.05 microns
and about 0.5 microns.
177. The method of claim 176, comprising sizing the liposomes to
have an average size of between about 0.05 microns and about 0.2
microns.
178. The method of claim 177, comprising sizing the liposomes to
have an average size selected from the group consisting of: between
about 0.05 microns and about 0.15 microns; and between about 0.05
microns and about 0.10 microns.
179. The method of any of claims 139-178, wherein the liposomal
composition comprises encapsulated aqueous solution and
non-encapsulated aqueous solution.
180. The method of claim 179, further comprising, after injecting
the lipid solution into the aqueous solution to form the liposomal
composition, removing the peptide epoxyketone compound from the
non-encapsulated aqueous solution.
181. The method of claim 180, wherein dialysis,
ultracentrifugation, or gel filtration is used for removing the
peptide epoxyketone compound from the non-encapsulated aqueous
solution.
182. The method of any of claims 139-181, further comprising, after
injecting the lipid solution into the aqueous solution to form the
liposomal composition, adjusting the aqueous solution to a pH of
between about pH 3.0 and about pH 7.0.
183. The method of any of claims 139-181, further comprising, after
injecting the lipid solution into the aqueous solution to form the
liposomal composition, adjusting the aqueous solution to a human
physiological pH.
184. The method of any of claims 139-183, further comprising, after
injecting the lipid solution into the aqueous solution to form the
liposomal composition, adding an agent to maintain isotonicity to
the aqueous solution.
185. The method of any of claims 139-184, wherein the peptide
epoxyketone compound is selected from the group consisting of
compound II, compound III, compound IV, and compound V.
186. The method of any of claims 139-184, wherein the peptide
epoxyketone compound is carfilzomib (compound V).
187. A pharmaceutical liposomal composition made by the method of
any of claims 139-186, the liposomal composition comprising
liposomes dispersed in the aqueous solution wherein the liposomes
comprise the peptide epoxyketone compound entrapped in the
liposomes.
188. A dry pharmaceutical composition formed by drying the
pharmaceutical liposomal composition of claim 187.
189. A method of treating multiple myeloma in a subject in need of
treatment, comprising: administering a therapeutically effective
amount of a pharmaceutical liposomal composition of any one of
claim 1-47, 87-90, 137, or 187.
190. The method of claim 189, further comprising simultaneous,
sequential, or separate administration of a therapeutically
effective amount of a chemotherapeutic agent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 61/673,017, filed 18 Jul. 2012, now pending,
and U.S. Provisional Application Ser. No. 61/794,603, filed 15 Mar.
2013, now pending, both of which applications are herein
incorporated by reference in their entireties.
TECHNICAL FIELD
[0002] The present invention relates generally to pharmaceutical
liposomal compositions comprising peptide epoxyketone compounds.
Aspects of the present invention include, but are not limited to,
methods for making stable pharmaceutical liposomal compositions
comprising peptide epoxyketone compounds, methods of using
pharmaceutical liposomal compositions, and dry pharmaceutical
compositions comprising peptide epoxyketone compounds made from the
pharmaceutical liposomal compositions.
BACKGROUND OF THE INVENTION
[0003] In eukaryotes, protein degradation is predominately mediated
through the ubiquitin pathway in which proteins targeted for
destruction are ligated to the 76 amino acid polypeptide ubiquitin.
Once targeted, ubiquitinated proteins then serve as substrates for
the 26S proteasome, a multicatalytic protease, which cleaves
proteins into short peptides through the action of its three major
proteolytic activities. While having a general function in
intracellular protein turnover, proteasome-mediated degradation
also plays a key role in many processes such as major
histocompatibility complex (MHC) class I presentation, apoptosis,
cell growth regulation, NF-.kappa.B activation, antigen processing,
and transduction of pro-inflammatory signals.
[0004] The 20S proteasome is a 700 kDa cylindrical-shaped
multicatalytic protease complex comprised of 28 subunits organized
into four rings. In yeast and other eukaryotes, 7 different .alpha.
subunits form the outer rings and 7 different .beta. subunits
comprise the inner rings. The .alpha. subunits serve as binding
sites for the 19S (PA700) and 11S (PA28) regulatory complexes, as
well as a physical barrier for the inner proteolytic chamber formed
by the two .beta. subunit rings. Thus, in vivo, the proteasome is
believed to exist as a 26S particle ("the 26S proteasome"). In vivo
experiments have shown that inhibition of the 20S form of the
proteasome can be readily correlated to inhibition of 26S
proteasome. Cleavage of amino-terminal prosequences of .beta.
subunits during particle formation exposes amino-terminal threonine
residues, which serve as the catalytic nucleophiles.
[0005] The subunits responsible for catalytic activity in
proteasomes thus possess an amino terminal nucleophilic residue,
and these subunits belong to the family of N-terminal nucleophile
(Ntn) hydrolases (where the nucleophilic N-terminal residue is, for
example, Cys, Ser, Thr, or other nucleophilic moieties). This
family includes, for example, penicillin G acylase (PGA),
penicillin V acylase (PVA), glutamine PRPP amidotransferase (GAT),
and bacterial glycosylasparaginase. In addition to the ubiquitously
expressed .beta. subunits, higher vertebrates also possess three
interferon-.gamma.-inducible .beta. subunits (LMP7, LMP2 and
MECL1), which replace their normal counterparts, .beta.5, .beta.1
and .beta.2 respectively, thus altering the catalytic activities of
the proteasome.
[0006] Through the use of different peptide substrates, three major
proteolytic activities have been defined for the eukaryote 20S
proteasome: chymotrypsin-like activity (CT-L), which cleaves after
large hydrophobic residues; trypsin-like activity (T-L), which
cleaves after basic residues; and caspase-like (C-L), which cleaves
after acidic residues. The major proteasome proteolytic activities
appear to be contributed by different catalytic sites, because
inhibitors, point mutations in .beta. subunits, and the exchange of
interferon-.gamma.-inducing .beta. subunits alter these activities
to various degrees.
[0007] There are several examples of small molecules that have been
used to inhibit proteasome activity and have been shown to be
effective against cancer, particularly multiple myeloma. However,
unlike the peptide epoxyketone compounds described herein, these
compounds generally lack the specificity, stability, or potency
necessary to explore and exploit the roles of the proteasome at the
cellular and molecular level, and thus maximize their therapeutic
activity.
SUMMARY OF THE INVENTION
[0008] The present invention generally relates to pharmaceutical
liposomal compositions comprising a peptide epoxyketone compound,
methods of making such liposomal compositions, methods of using
such liposomal compositions, dry pharmaceutical compositions
comprising a peptide epoxyketone compound, and methods of making
and using such dry pharmaceutical compositions.
[0009] In one aspect, the present invention relates to
pharmaceutical liposomal compositions. In some embodiments, the
pharmaceutical liposomal compositions comprise liposome entrapped
peptide epoxyketone compound. Such pharmaceutical compositions
typically comprise an aqueous solution comprising liposomes,
wherein the liposomes comprise between about 0.5 wt. % and about 50
wt. % of a peptide epoxyketone compound, and between about 99.5 wt.
% and about 50 wt. % total lipids (weight ratio of peptide
epoxyketone compound:total lipids of between about 0.005:0.995 and
about 0.5:0.5).
[0010] In preferred embodiments the total lipids comprise a
phospholipid, for example, L-.alpha.-phosphatidylcholine,
1,2-distearoyl-sn-glycero-3-phosphocholine,
1,2-dipalmitoyl-sn-glycero-3-phosphocholine,
1,2-Distearoyl-sn-glycero-3-phospho-rac-(1-glycerol),
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, sphingomyelin,
1,2-distearoyl-sn-glycero-3-phosphoethanolamine, as well as
combinations thereof. Total lipids can further comprise, for
example, a hydrophilic polymer-derivatized lipid, and/or a
cholesterol or cholesterol derivative.
[0011] The liposomes of the liposomal compositions comprising
peptide epoxyketone compounds typically have an average size of
between about 0.05 microns and about 0.5 microns.
[0012] In some embodiments of the present invention, the liposomal
compositions comprise liposomes comprising the peptide epoxyketone
compound and a solubilizing agent in an internal aqueous core of
the liposomes. In some embodiments, the solubilizing agent is a
compound (e.g., a cyclodextrin), and the liposomes of the liposomal
composition comprise the peptide epoxyketone compound complexed
with the compound (e.g., a cyclodextrin) in the internal aqueous
core of the liposomes.
[0013] The pharmaceutical liposomal compositions of the present
invention can also include one or more excipients.
[0014] In other aspects, the present invention relates to dry
pharmaceutical compositions comprising peptide epoxyketone
compounds. Such dry pharmaceutical compositions are typically made
by dehydration of the pharmaceutical liposomal compositions
described here.
[0015] In yet further aspects, the present invention relates to
methods of making the pharmaceutical liposomal compositions
described herein. One method of making a pharmaceutical liposomal
composition comprises preparing a dried film comprising total
lipids, and rehydrating the dried film with an aqueous solution
comprising a peptide epoxyketone compound to form a liposomal
composition comprising liposomes dispersed in aqueous solution.
Typically the aqueous solution comprises a peptide epoxyketone
compound and a solubilizing agent. Another method of making a
pharmaceutical liposomal composition comprises preparing a lipid
solution comprising total lipids and a solvent, and injecting the
lipid solution into an aqueous solution comprising a peptide
epoxyketone compound. Typically the aqueous solution comprises a
peptide epoxyketone compound and a solubilizing agent.
[0016] In some embodiments, peptide epoxyketone compounds not
encapsulated in liposomes are removed from the pharmaceutical
liposomal compositions.
[0017] In other aspects, the present invention relates to
pharmaceutical liposomal compositions comprising peptide
epoxyketone compounds made by the methods of the invention, dry
pharmaceutical compositions made therefrom, as well as
reconstituted liposomal compositions comprising peptide epoxyketone
compounds made from the dry pharmaceutical compositions.
[0018] In further aspects, the present invention relates to methods
of treating a disease or condition in a subject in need of
treatment, comprising administering a therapeutically effective
amount of a pharmaceutical liposomal composition comprising
liposomes comprising a peptide epoxyketone compound. In some
embodiments the methods of treating further comprise simultaneous,
sequential, or separate administration of a therapeutically
effective amount of another therapeutic agent, for example, a
chemotherapeutic agent, a cytokine, a steroid, an immunotherapeutic
agent, or combinations thereof.
[0019] These and other embodiments of the present invention will
readily occur to those of ordinary skill in the art in view of the
disclosure herein.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 presents specific experimental formulations and HPLC
characterization data of selected, exemplary liposomal compositions
(Compositions A to G; Table 2A), which were used to generate
associated data presented in the Examples and Tables herein. The
nominal concentrations of exemplary liposomal Compositions A to G
are presented in Table 2A. Note that the drug to lipid ratios in
FIG. 1 (Drug:Lipid Ratio (%)) were calculated by taking the weight
of drug (carfilzomib) divided by the weight of drug plus weight of
phospholipid; however, the more conventional calculation is a ratio
of the weight of drug to the weight of total lipids (e.g.
phospholipid, hydrophilic polymer-derivatized lipid, cholesterol;
see, e.g., Table 3). The more conventional calculation of
Drug:Total Lipid Ratio for the specific experimental formulations
in FIG. 1 are presented in Table 2B. The columns for the HPLC
characterization data are as follows: "n" is the number of
replicate samples assayed; "% Diff" is the percent difference
between the theoretical drug concentration and the average
experimental drug concentration; "SD" is the standard deviation of
the experimentally determined drug concentration; and "% RSD" is
the percent relative standard deviation of the experimentally
determined drug concentration. The rows of "Pooled" data in FIG. 1
represent instances where two separate batches of a particular
liposomal composition were combined into a single batch liposomal
composition (see, e.g., "6005-45A/B Pooled" represents a liposomal
Composition C prepared by combining Composition C (6005-45A) and
Composition C (6005-45B)).
[0021] FIG. 2A presents data related to pharmacodynamic responses
in BALB/C mice to different compositions of carfilzomib. In the
figure, the vertical axis is the percent (%) enzymatic activity
relative to vehicle, wherein the enzymatic activity corresponds to
proteasome CT-L activity in whole blood (primarily erythrocytes).
Three groups of data are presented on the horizontal axis as
follows. The first group, represented in the figure as
cross-hatched bars, presents data for an injectable composition of
carfilzomib (CFZ) formulated in 10% sulfobutylether beta
cyclodextrin (SBE-.beta.-CD, also referred to herein as SBE-B-CD),
and 10 mM Citrate, pH 3.5 (non-liposomal): the first bar presents
control data for the placebo vehicle without carfilzomib, the
second bar presents data for CFZ SBE-B-CD at 1 hour, the third bar
presents data for CFZ SBE-B-CD at 8 hours, and the fourth bar
presents data for CFZ SBE-B-CD at 24 hours. The second group,
represented in the figure as diagonal-lined bars, presents data for
a liposomal composition of carfilzomib (liposomes comprising CFZ
(L-CFZ, Composition C)): the first bar presents control data for
the liposomal vehicle without carfilzomib, the second bar presents
data for the carfilzomib liposomal composition at 1 hour, the third
bar presents data for the carfilzomib liposomal composition at 8
hours, and the fourth bar presents data for the carfilzomib
liposomal composition at 24 hours. The third group, represented in
the figure as white, outlined bars, presents data for a pegylated
liposomal composition of carfilzomib (pegylated liposomes
comprising (pL-CFZ, Composition E)): the first bar presents control
data for the pegylated liposomal vehicle without carfilzomib, the
second bar presents data for the carfilzomib pegylated liposomal
composition at 1 hour, the third bar presents data for the
carfilzomib pegylated liposomal composition at 8 hours, and the
fourth bar presents data for the carfilzomib pegylated liposomal
composition at 24 hours. Error bars are represented
unidirectionally.
[0022] FIG. 2B presents data related to pharmacodynamic responses
in BALB/C mice to different compositions of carfilzomib. In the
figure, the vertical axis is the percent (%) enzymatic activity
relative to vehicle, wherein the enzymatic activity corresponds to
proteasome CT-L activity in adrenal tissue. Three groups of data
are presented on the horizontal axis as follows. The first group,
represented in the figure as cross-hatched bars, presents data for
injectable CFZ SBE-B-CD (non-liposomal): the first bar presents
control data for the vehicle without carfilzomib, the second bar
presents data for CFZ SBE-B-CD at 1 hour, the third bar presents
data for CFZ SBE-B-CD at 8 hours, and the fourth bar presents data
for CFZ SBE-B-CD at 24 hours. The second group, represented in the
figure as diagonal-lined bars, presents data for a liposomal
composition of carfilzomib (liposomes comprising CFZ (L-CFZ,
Composition C)): the first bar presents control data for the
liposomal vehicle without carfilzomib, the second bar presents data
for the carfilzomib liposomal composition at 1 hour, the third bar
presents data for the carfilzomib liposomal composition at 8 hours,
and the fourth bar presents data for the carfilzomib liposomal
composition at 24 hours. The third group, represented in the figure
as white, outlined bars, presents data for a pegylated liposomal
composition of carfilzomib (pegylated liposomes comprising (pL-CFZ,
Composition E)): the first bar presents control data for the
pegylated liposomal vehicle without carfilzomib, the second bar
presents data for the carfilzomib pegylated liposomal composition
at 1 hour, the third bar presents data for the carfilzomib
pegylated liposomal composition at 8 hours, and the fourth bar
presents data for the carfilzomib pegylated liposomal composition
at 24 hours. Error bars are represented unidirectionally.
[0023] FIG. 2C presents data related to pharmacodynamic responses
in BALB/C mice to different compositions of carfilzomib. In the
figure, the vertical axis is the percent (%) enzymatic activity
relative to vehicle, wherein the enzymatic activity corresponds to
proteasome CT-L activity in liver tissue. Three groups of data are
presented on the horizontal axis as follows. The first group,
represented in the figure as cross-hatched bars, presents data for
injectable CFZ SBE-B-CD (non-liposomal): the first bar presents
control data for the vehicle without carfilzomib, the second bar
presents data for the carfilzomib SBE-B-CD composition at 1 hour,
the third bar presents data for the carfilzomib composition at 8
hours, and the fourth bar presents data for the carfilzomib
composition at 24 hours. The second group, represented in the
figure as diagonal-lined bars, presents data for a liposomal
composition of carfilzomib (liposomes comprising CFZ (L-CFZ,
Composition C)): the first bar presents control data for the
liposomal vehicle without carfilzomib, the second bar presents data
for the carfilzomib liposomal composition at 1 hour, the third bar
presents data for the carfilzomib liposomal composition at 8 hours,
and the fourth bar presents data for the carfilzomib liposomal
composition at 24 hours. The third group, represented in the figure
as white, outlined bars, presents data for a pegylated liposomal
composition of carfilzomib (pegylated liposomes comprising (pL-CFZ,
Composition E)): the first bar presents control data for the
pegylated liposomal vehicle without carfilzomib, the second bar
presents data for the carfilzomib pegylated liposomal composition
at 1 hour, the third bar presents data for the carfilzomib
pegylated liposomal composition at 8 hours, and the fourth bar
presents data for the carfilzomib pegylated liposomal composition
at 24 hours. Error bars are represented unidirectionally.
[0024] FIG. 2D presents data related to pharmacodynamic responses
in BALB/C mice to different compositions of carfilzomib. In the
figure, the vertical axis is the percent (%) enzymatic activity
relative to vehicle, wherein the enzymatic activity corresponds to
proteasome CT-L activity in heart tissue. Three groups of data are
presented on the horizontal axis as follows. The first group,
represented in the figure as cross-hatched bars, presents data for
injectable CFZ SBE-B-CD (non-liposomal): the first bar presents
control data for the vehicle without carfilzomib, the second bar
presents data for CFZ SBE-B-CD at 1 hour, the third bar presents
data for CFZ SBE-B-CD at 8 hours, and the fourth bar presents data
for CFZ SBE-B-CD at 24 hours. The second group, represented in the
figure as diagonal-lined bars, presents data for a liposomal
composition of carfilzomib (liposomes comprising CFZ (L-CFZ,
Composition C)): the first bar presents control data for the
liposomal vehicle without carfilzomib, the second bar presents data
for the carfilzomib liposomal composition at 1 hour, the third bar
presents data for the carfilzomib liposomal composition at 8 hours,
and the fourth bar presents data for the carfilzomib liposomal
composition at 24 hours. The third group, represented in the figure
as white, outlined bars, presents data for a pegylated liposomal
composition of carfilzomib (pegylated liposomes comprising (pL-CFZ,
Composition E)): the first bar presents control data for the
pegylated liposomal vehicle without carfilzomib, the second bar
presents data for the carfilzomib pegylated liposomal composition
at 1 hour, the third bar presents data for the carfilzomib
pegylated liposomal composition at 8 hours, and the fourth bar
presents data for the carfilzomib pegylated liposomal composition
at 24 hours. Error bars are represented unidirectionally.
[0025] FIG. 3A presents data related to pharmacodynamic responses
in BALB/C mice to different compositions of carfilzomib. In the
figure, the vertical axis (CT-L activity) is the percent (%)
enzymatic activity relative to a corresponding vehicle without
carfilzomib (CFZ), wherein the enzymatic activity corresponds to
proteasome CT-L activity in whole blood (primarily erythrocytes).
The horizontal axis is the time in hours (Hour). Four groups of
data are presented. The first group (open squares containing an X)
presents data for an injectable composition of carfilzomib (CFZ)
formulated in 10% sulfobutylether beta cyclodextrin (SBE-B-CD), and
10 mM Citrate, pH 3.5, (non-liposomal) administered at 5 mg/kg with
data points at 0, 1, 4, 6, 8, and 24 hours. The second group (open
circles containing an X) presents data for an injectable
composition of carfilzomib (CFZ) formulated in 10% sulfobutylether
beta cyclodextrin (SBE-B-CD), and 10 mM Citrate, pH 3.5,
(non-liposomal) administered at 10 mg/kg with data points at 0, 1,
8, and 24 hours. The third group (solid squares) presents data for
a pegylated liposomal composition of carfilzomib wherein the
aqueous core of the pegylated liposomes comprises carfilzomib and
SBE-B-CD (ap-L11) administered at 5 mg/kg with data points at 0, 1,
4, 6, and 24 hours. The fourth group (solid circles) presents data
for a pegylated liposomal composition of carfilzomib wherein the
aqueous core of the pegylated liposomes comprises carfilzomib and
SBE-B-CD (ap-L11) administered at 15 mg/kg with data points at 0,
1, 4, 6, and 24 hours. Error bars are represented
unidirectionally.
[0026] FIG. 3B presents data related to pharmacodynamic responses
in BALB/C mice to different compositions of carfilzomib. In the
figure, the vertical axis (CT-L activity) is the percent (%)
enzymatic activity relative to a corresponding vehicle without
carfilzomib (CFZ), wherein the enzymatic activity corresponds to
proteasome CT-L activity in heart tissue. The horizontal axis is
the time in hours (Hour). Four groups of data are presented. The
first group (open squares containing an X) presents data for an
injectable composition of carfilzomib (CFZ) formulated in 10%
sulfobutylether beta cyclodextrin (SBE-B-CD), and 10 mM Citrate, pH
3.5, (non-liposomal) administered at 5 mg/kg with data points at 0,
1, 4, 6, 8, and 24 hours. The second group (open circles containing
an X) presents data for an injectable composition of carfilzomib
(CFZ) formulated in 10% sulfobutylether beta cyclodextrin
(SBE-B-CD), and 10 mM Citrate, pH 3.5, (non-liposomal) administered
at 10 mg/kg with data points at 0, 1, 8, and 24 hours. The third
group (solid squares) presents data for a pegylated liposomal
composition of carfilzomib wherein the aqueous core of the
pegylated liposomes comprises carfilzomib and SBE-B-CD (ap-L11)
administered at 5 mg/kg with data points at 0, 1, 4, 6, and 24
hours. The fourth group (solid circles) presents data for a
pegylated liposomal composition of carfilzomib wherein the aqueous
core of the pegylated liposomes comprises carfilzomib and SBE-B-CD
(ap-L11) administered at 15 mg/kg with data points at 0, 1, 4, 6,
and 24 hours. Error bars are represented unidirectionally.
[0027] FIG. 3C presents data related to pharmacodynamic responses
in BALB/C mice to different compositions of carfilzomib. In the
figure, the vertical axis (CT-L activity) is the percent (%)
enzymatic activity relative to a corresponding vehicle without
carfilzomib (CFZ), wherein the enzymatic activity corresponds to
proteasome CT-L activity in liver tissue. The horizontal axis is
the time in hours (Hour). Four groups of data are presented. The
first group (open squares containing an X) presents data for an
injectable composition of carfilzomib (CFZ) formulated in 10%
sulfobutylether beta cyclodextrin (SBE-B-CD), and 10 mM Citrate, pH
3.5, (non-liposomal) administered at 5 mg/kg with data points at 0,
1, 4, 6, 8, and 24 hours. The second group (open circles containing
an X) presents data for an injectable composition of carfilzomib
(CFZ) formulated in 10% sulfobutylether beta cyclodextrin
(SBE-B-CD), and 10 mM Citrate, pH 3.5, (non-liposomal) administered
at 10 mg/kg with data points at 0, 1, 8, and 24 hours. The third
group (solid squares) presents data for a pegylated liposomal
composition of carfilzomib wherein the aqueous core of the
pegylated liposomes comprises carfilzomib and SBE-B-CD (ap-L11)
administered at 5 mg/kg with data points at 0, 1, 4, 6, and 24
hours. The fourth group (solid circles) presents data for a
pegylated liposomal composition of carfilzomib wherein the aqueous
core of the pegylated liposomes comprises carfilzomib and SBE-B-CD
(ap-L11) administered at 15 mg/kg with data points at 0, 1, 4, 6,
and 24 hours. Error bars are represented unidirectionally.
[0028] FIG. 3D presents data related to pharmacodynamic responses
in BALB/C mice to different compositions of carfilzomib. In the
figure, the vertical axis (CT-L activity) is the percent (%)
enzymatic activity relative to a corresponding vehicle without
carfilzomib (CFZ), wherein the enzymatic activity corresponds to
proteasome CT-L activity in adrenal tissue. The horizontal axis is
the time in hours (Hour). Four groups of data are presented. The
first group (open squares containing an X) presents data for an
injectable composition of carfilzomib (CFZ) formulated in 10%
sulfobutylether beta cyclodextrin (SBE-B-CD), and 10 mM Citrate, pH
3.5, (non-liposomal) administered at 5 mg/kg with data points at 0,
1, 4, 6, 8, and 24 hours. The second group (open circles containing
an X) presents data for an injectable composition of carfilzomib
(CFZ) formulated in 10% sulfobutylether beta cyclodextrin
(SBE-B-CD), and 10 mM Citrate, pH 3.5, (non-liposomal) administered
at 10 mg/kg with data points at 0, 1, 8, and 24 hours. The third
group (solid squares) presents data for a pegylated liposomal
composition of carfilzomib wherein the aqueous core of the
pegylated liposomes comprises carfilzomib and SBE-B-CD (ap-L11)
administered at 5 mg/kg with data points at 0, 1, 4, 6, and 24
hours. The fourth group (solid circles) presents data for a
pegylated liposomal composition of carfilzomib wherein the aqueous
core of the pegylated liposomes comprises carfilzomib and SBE-B-CD
(ap-L11) administered at 15 mg/kg with data points at 0, 1, 4, 6,
and 24 hours. Error bars are represented unidirectionally.
[0029] FIG. 4 presents data related to the circulation half-life in
BALB/C mice of different compositions of carfilzomib. In the
figure, the vertical axis is the concentration of carfilzomib in
umol/L (Concentration (umol/L)), and the horizontal axis is the
time in minutes (Time (min)). The line with open squares containing
an X corresponds to administration of 5 mg/kg of an injectable
carfilzomib SBE-B-CD composition (non-liposomal). The line with
solid squares corresponds to administration of 5 mg/kg of apL11, a
pegylated liposomal composition of carfilzomib wherein the aqueous
core of the pegylated liposomes comprises carfilzomib and SBE-B-CD.
The line with solid circles corresponds to administration of 15
mg/kg of apL11, a pegylated liposomal composition of carfilzomib
wherein the aqueous core of the pegylated liposomes comprises
carfilzomib and SBE-B-CD. Error bars are represented
bidirectionally.
[0030] FIG. 5A presents data related to pharmacodynamic responses
in BALB/C mice to different compositions of carfilzomib. In the
figure, the vertical axis (CT-L Activity) is the percent (%)
enzymatic activity relative to a corresponding vehicle without
carfilzomib (CFZ), wherein the enzymatic activity corresponds to
proteasome CT-L activity in whole blood (primarily erythrocytes).
The horizontal axis is the time in hours (Hour). Two groups of data
are presented. The first group (open circles) presents data for an
injectable composition of carfilzomib (CFZ) formulated in 10%
sulfobutylether beta cyclodextrin (SBE-B-CD), and 10 mM Citrate, pH
3.5, (non-liposomal) administered at 10 mg/kg with data points at
0, 1, 8, and 24 hours. The second group (solid squares) presents
data for a liposomal composition of carfilzomib comprising
liposomes comprising entrapped carfilzomib (pL-6) administered at
15 mg/kg with data points at 0, 1, 4, 6, and 24 hours. Error bars
are represented bidirectionally.
[0031] FIG. 5B presents data related to pharmacodynamic responses
in BALB/C mice to different compositions of carfilzomib. In the
figure, the vertical axis (CT-L Activity) is the percent (%)
enzymatic activity relative to a corresponding vehicle without
carfilzomib (CFZ), wherein the enzymatic activity corresponds to
proteasome CT-L activity in heart tissue. The horizontal axis is
the time in hours (Hour). Two groups of data are presented. The
first group (open circles) presents data for an injectable
composition of carfilzomib (CFZ) formulated in 10% sulfobutylether
beta cyclodextrin (SBE-B-CD), and 10 mM Citrate, pH 3.5,
(non-liposomal) administered at 10 mg/kg with data points at 0, 1,
8, and 24 hours. The second group (solid squares) presents data for
a liposomal composition of carfilzomib comprising liposomes
comprising entrapped carfilzomib (pL-6) administered at 15 mg/kg
with data points at 0, 1, 4, 6, and 24 hours. Error bars are
represented bidirectionally.
[0032] FIG. 5C presents data related to pharmacodynamic responses
in BALB/C mice to different compositions of carfilzomib. In the
figure, the vertical axis (CT-L Activity) is the percent (%)
enzymatic activity relative to a corresponding vehicle without
carfilzomib (CFZ), wherein the enzymatic activity corresponds to
proteasome CT-L activity in liver tissue. The horizontal axis is
the time in hours (Hour). Two groups of data are presented. The
first group (open circles) presents data for an injectable
composition of carfilzomib (CFZ) formulated in 10% sulfobutylether
beta cyclodextrin (SBE-B-CD), and 10 mM Citrate, pH 3.5,
(non-liposomal) administered at 10 mg/kg with data points at 0, 1,
8, and 24 hours. The second group (solid squares) presents data for
a liposomal composition of carfilzomib comprising liposomes
comprising entrapped carfilzomib (pL-6) administered at 15 mg/kg
with data points at 0, 1, 4, 6, and 24 hours. Error bars are
represented bidirectionally.
[0033] FIG. 5D presents data related to pharmacodynamic responses
in BALB/C mice to different compositions of carfilzomib. In the
figure, the vertical axis (CT-L Activity) is the percent (%)
enzymatic activity relative to a corresponding vehicle without
carfilzomib (CFZ), wherein the enzymatic activity corresponds to
proteasome CT-L activity in adrenal tissue. The horizontal axis is
the time in hours (Hour). Two groups of data are presented. The
first group (open circles) presents data for an injectable
composition of carfilzomib (CFZ) formulated in 10% sulfobutylether
beta cyclodextrin (SBE-B-CD), and 10 mM Citrate, pH 3.5,
(non-liposomal) administered at 10 mg/kg with data points at 0, 1,
8, and 24 hours. The second group (solid squares) presents data for
a liposomal composition of carfilzomib comprising liposomes
comprising entrapped carfilzomib (pL-6) administered at 15 mg/kg
with data points at 0, 1, 4, 6, and 24 hours. Error bars are
represented bidirectionally.
[0034] FIG. 6 presents data related to the circulation half-life in
BALB/C mice of different compositions of carfilzomib. In the
figure, the vertical axis is the concentration of carfilzomib in
umol/L (Concentration (umol/L)), and the horizontal axis is the
time post dose in minutes (Time Post Dose (min)). The line with
open circles corresponds to administration of 5 mg/kg of an
injectable carfilzomib SBE-B-CD composition (non-liposomal). The
line with solid squares corresponds to administration of 15 mg/kg
of a liposomal composition of carfilzomib comprising liposomes
comprising entrapped carfilzomib (pL-6). Error bars are represented
unidirectionally.
[0035] FIG. 7 presents data related to the dosing frequency of
different compositions of carfilzomib in a mouse xenograft tumor
model. In the figure, the vertical axis is tumor volume in mm.sup.3
(Tumor Volume (mm.sup.3)), and the horizontal axis is days
post-tumor challenge (Days). The line (top line in the figure at 30
days) with open circles correspond to once-weekly administration of
vehicle (liposomes comprising 12.5 mg/mL EPC, 3.1 mg/mL
Cholesterol, 1.2 mg/mL mPEG-DSPE, with no carfilzomib); the line
with open squares corresponds to 5 mg/kg of a non-liposomal
carfilzomib SBE-B-CD composition (non-liposomal) administered on
days 1 and 2 of each week; the line with solid circles corresponds
to administration of pL-6=2 mg/mL CFZ, 12.5 mg/mL Sphingomylin, 3.2
mg/mL cholesterol, 1.3 mg/mL mPEG-DSPE providing a dose of 10 mg/kg
of carfilzomib administered on days 1 and 2 of each week. The line
with open triangles corresponds to administration of pL-6=2 mg/mL
CFZ, 12.5 mg/mL Sphingomylin, 3.2 mg/mL cholesterol, 1.3 mg/mL
mPEG-DSPE providing a dose of 15 mg/kg of carfilzomib administered
once weekly. This figure presents data up to day 31. Error bars are
represented unidirectionally.
[0036] FIG. 8 corresponds to FIG. 7, with the exception that FIG. 8
presents data up to day 38 (i.e., two additional time points
relative to FIG. 7). Error bars are represented
unidirectionally.
[0037] FIG. 9 presents data related to dose and dosing frequency in
a mouse xenograft tumor model for a liposomal composition of
carfilzomib comprising liposomes comprising entrapped carfilzomib
(Composition C; 2 mg/ml CFZ, 12.5 mg/ml EPC). In the figure, the
vertical axis is tumor volume in mm.sup.3 (Tumor Volume
(mm.sup.3)), and the horizontal axis is days post-tumor challenge
(Days Post Tumor Challenge). The line (top line in the figure at 30
days) with solid circles correspond to once-weekly administration
(QW) of vehicle (empty liposomes comprising 12.5 mg/mL EPC); the
line with solid squares corresponds to 5 mg/kg of a non-liposomal
CFZ composition administered on days 1 and 2 of each week
(QD.times.2). The line with open diamonds corresponds to 5 mg/kg of
liposomal Composition C administered on days 1 and 2 of each week
(QD.times.2). The line with open triangles corresponds to 10 mg/kg
of liposomal Composition C administered on days 1 and 2 of each
week (QD.times.2). The line with open circles corresponds to 15
mg/kg of liposomal Composition C administered once weekly (QW).
Error bars are represented unidirectionally.
[0038] FIG. 10 presents data related to dose and dosing frequency
in a mouse xenograft tumor model for a pegylated liposomal
composition of carfilzomib comprising liposomes comprising
entrapped carfilzomib (Composition G; 2 mg/ml CFZ, 12.5 mg/ml EPC,
1.3 mg/ml mPEG-DSPE, 3.1 mg/ml cholesterol). In the figure, the
vertical axis is tumor volume in mm.sup.3 (Tumor Volume
(mm.sup.3)), and the horizontal axis is days post-tumor challenge.
The line (top line in the figure at 40 days) with open circles
containing an X correspond to once-weekly administration of vehicle
(empty liposomes comprising 12.5 mg/mL EPC, 10% mPEG, 25%
Cholesterol); the line with open triangles corresponds to 5 mg/kg
of a non-liposomal CFZ composition administered on days 1 and 2 of
each week. The line with open squares corresponds to 10 mg/kg of
liposomal Composition G administered on days 1 and 2 of each week.
The line with solid circles corresponds to 15 mg/kg of liposomal
Composition G administered once week. Error bars are represented
unidirectionally.
DETAILED DESCRIPTION OF THE INVENTION
[0039] All patents, publications, and patent applications cited in
this specification are herein incorporated by reference as if each
individual patent, publication, or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety for all purposes.
1.0.0 DEFINITIONS
[0040] It is to be understood that the terminology used herein is
for the purpose of describing particular embodiments only, and is
not intended to be limiting. As used in this specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a lipid" includes one or more
lipids, or mixtures of lipids; reference to "a phospholipid"
includes one or more lipids, or mixtures of phospholipids;
reference to "a cholesterol or cholesterol derivative" includes one
or more cholesterol or cholesterol derivative, or mixtures of a
cholesterol and a cholesterol derivative; reference to "a
hydrophilic polymer-derivatized lipid" includes one or more a
hydrophilic polymer-derivatized lipids, or mixtures of a
hydrophilic polymer-derivatized lipids; reference to "a hydrophilic
polymer" includes one or more hydrophilic polymers, or mixtures of
hydrophilic polymers; reference to "a drug" includes one or more
drugs, and the like.
[0041] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. Although
other methods and materials similar, or equivalent, to those
described herein can be used in the practice of the present
invention, the preferred materials and methods are described
herein.
[0042] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions set out below.
[0043] The term "enzyme" as used herein refers to any partially or
wholly proteinaceous molecule that carries out a chemical reaction
in a catalytic manner. Such enzymes can be native enzymes, fusion
enzymes, proenzymes, apoenzymes, denatured enzymes, farnesylated
enzymes, ubiquitinated enzymes, fatty acylated enzymes,
gerangeranylated enzymes, GPI-linked enzymes, lipid-linked enzymes,
prenylated enzymes, naturally-occurring or artificially generated
mutant enzymes, enzymes with side chain or backbone modifications,
enzymes having leader sequences, and enzymes complexed with
non-proteinaceous material, such as proteoglycans and
proteoliposomes. Enzymes can be made by any means, including
natural expression, promoted expression, cloning, various
solution-based and solid-based peptide syntheses, and similar
methods known to those skilled in the art.
[0044] The term "C.sub.x-y alkyl" as used herein refers to
substituted or unsubstituted saturated hydrocarbon groups,
including straight-chain alkyl and branched-chain alkyl groups that
contain from x to y carbons in the chain, including haloalkyl
groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc.
C.sub.0 alkyl indicates a hydrogen where the group is in a terminal
position, a bond if internal. The terms "C.sub.2-y alkenyl" and
"C.sub.2-y alkynyl" refer to substituted or unsubstituted
unsaturated aliphatic groups analogous in length and possible
substitution to the alkyls, but that contain at least one double or
triple bond respectively.
[0045] The term "alkoxy" as used herein refers to an alkyl group
having an oxygen attached thereto. Representative alkoxy groups
include methoxy, ethoxy, propoxy, tert-butoxy, and the like.
[0046] The term "ether" as used herein refers to two hydrocarbons
covalently linked by an oxygen. Accordingly, the substituent of an
alkyl that renders that alkyl an ether is or resembles an
alkoxy.
[0047] The term "C.sub.1-6 alkoxyalkyl" as used herein refers to a
C.sub.1-6 alkyl group substituted with an alkoxy group, thereby
forming an ether.
[0048] The term "C.sub.1-6 aralkyl" as used herein refers to a
C.sub.1-6 alkyl group substituted with an aryl group.
[0049] The terms "amine" and "amino" as used herein are
art-recognized and refer to both unsubstituted and substituted
amines and salts thereof, e.g., a moiety that can be represented by
the general formulae:
##STR00001##
[0050] wherein R.sup.9, R.sup.10 and R.sup.10' each independently
represent a hydrogen, an alkyl, an alkenyl,
--(CH.sub.2).sub.m--R.sup.8, or R.sup.9 and R.sup.10 taken together
with the N atom to which they are attached complete a heterocycle
having from 4 to 8 atoms in the ring structure; R.sup.8 represents
an aryl, a cycloalkyl, a cycloalkenyl, a heterocyclyl or a
polycyclyl; and m is zero or an integer from 1 to 8. In preferred
embodiments, only one of R.sup.9 or R.sup.10 can be a carbonyl,
e.g., R.sup.9, R.sup.10, and the nitrogen together do not form an
imide. In even more preferred embodiments, R.sup.9 and R.sup.10
(and optionally R.sup.10') each independently represent a hydrogen,
an alkyl, an alkenyl, or --(CH.sub.2).sub.m--R.sup.8. In certain
embodiments, the amino group is basic, meaning the protonated form
has a pK.sub.a.gtoreq.7.00.
[0051] The terms "amide" and "amido" are art-recognized as
referring to an amino-substituted carbonyl and including a moiety
that can be represented by the general formula:
##STR00002##
[0052] wherein R.sup.9, R.sup.10 are as defined above. Preferred
embodiments of the amide does not include imides that can be
unstable.
[0053] The term "aryl" as used herein refers to 5-membered,
6-membered, and 7-membered substituted or unsubstituted single-ring
aromatic groups in which each atom of the ring is carbon. The term
"aryl" also includes polycyclic ring systems having two or more
cyclic rings in which two or more carbons are common to two
adjoining rings, wherein at least one of the rings is aromatic,
e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,
cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl
groups include benzene, naphthalene, phenanthrene, phenol, aniline,
and the like.
[0054] The terms "carbocycle" and "carbocyclyl" as used herein
refer to a non-aromatic substituted or unsubstituted ring in which
each atom of the ring is carbon. The terms "carbocycle" and
"carbocyclyl" also include polycyclic ring systems having two or
more cyclic rings in which two or more carbons are common to two
adjoining rings wherein at least one of the rings is carbocyclic,
e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,
cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
[0055] The term "carbonyl" as used herein is art-recognized and
refers to moieties as can be represented by the general
formula:
##STR00003##
wherein X is a bond or represents an oxygen or a sulfur, and
R.sup.11 represents a hydrogen, an alkyl, an alkenyl,
--(CH.sub.2).sub.m--R.sup.8, or a pharmaceutically acceptable salt;
R.sup.11' represents a hydrogen, an alkyl, an alkenyl, or
--(CH.sub.2).sub.m--R.sup.8, where m and R.sup.8 are as defined
below. Where X is an oxygen and R.sup.11 or R.sup.11' is not
hydrogen, the formula represents an "ester." Where X is an oxygen
and R.sup.11 is a hydrogen, the formula represents a "carboxylic
acid."
[0056] The term "C.sub.1-6 heteroaralkyl" as used herein refers to
a C.sub.1-6 alkyl group substituted with a heteroaryl group.
[0057] The term "heteroaryl" as used herein refers to substituted
or unsubstituted aromatic 5-membered to 7-membered ring structures,
more preferably 5-membered to 6-membered rings, whose ring
structures include one to four heteroatoms. The term "heteroaryl"
also includes polycyclic ring systems having two or more cyclic
rings in which two or more carbons are common to two adjoining
rings, wherein at least one of the rings is heteroaromatic, e.g.,
the other cyclic rings can be cycloalkyls, cycloalkenyls,
cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl
groups include, for example, pyrrole, furan, thiophene, imidazole,
oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine,
pyridazine, pyrimidine, and the like.
[0058] The term "heteroatom" as used herein refers to an atom of
any element other than carbon or hydrogen. Preferred heteroatoms
are nitrogen, oxygen, phosphorus, and sulfur.
[0059] The terms "heterocyclyl" and "heterocyclic group" as used
herein refer to substituted or unsubstituted non-aromatic
3-membered to 10-membered ring structures, more preferably
3-membered to 7-membered rings, whose ring structures include one
to four heteroatoms. The term terms "heterocyclyl" or "heterocyclic
group" also include polycyclic ring systems having two or more
cyclic rings in which two or more carbons are common to two
adjoining rings, wherein at least one of the rings is heterocyclic,
e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,
cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
Heterocyclyl groups include, for example, piperidine, piperazine,
pyrrolidine, morpholine, lactones, lactams, and the like.
[0060] The term "C.sub.1-6hydroxyalkyl" as used herein refers to a
C.sub.1-6alkyl group substituted with a hydroxy group.
[0061] The term "thioether" as used herein refers to an alkyl group
having a sulfur moiety attached thereto. In preferred embodiments,
the "thioether" is represented by --S-- alkyl. Representative
thioether groups include methylthio, ethylthio, and the like.
[0062] The term "substituted" as used herein refers to moieties
having substituents replacing a hydrogen on one or more carbons of
the backbone. The terms "substitution" or "substituted with"
include the implicit proviso that such substitution is in
accordance with permitted valence of the substituted atom and the
substituent, and that the substitution results in a stable
compound, e.g., which does not spontaneously undergo transformation
such as by rearrangement, cyclization, elimination, etc. As used
herein, the term "substituted" is contemplated to include all
permissible substituents of organic compounds. In a broad aspect,
the permissible substituents include acyclic and cyclic, branched
and unbranched, carbocyclic and heterocyclic, aromatic and
non-aromatic substituents of organic compounds. The permissible
substituents can be one or more and the same or different for
appropriate organic compounds. For purposes of this invention, the
heteroatoms such as nitrogen may have hydrogen substituents and/or
any permissible substituents of organic compounds described herein
that satisfy the valences of the heteroatoms. Substituents can
include, for example, a halogen, a hydroxyl, a carbonyl (such as a
carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl
(such as a thioester, a thioacetate, or a thioformate), an alkoxyl,
a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino,
an amido, an amidine, an imine, a cyano, a nitro, an azido, a
sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a
sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic
or heteroaromatic moiety. It will be understood by those skilled in
the art that the moieties substituted on the hydrocarbon chain can
themselves be substituted, if appropriate.
[0063] The term "inhibitor" as used herein refers to a compound
that blocks or reduces an activity of an enzyme (e.g., inhibition
of proteolytic cleavage of standard fluorogenic peptide substrates
such as suc-LLVY-AMC, Boc-LLR-AMC and Z-LLE-AMC, inhibition of
various catalytic activities of the 20S proteasome). An inhibitor
can act with competitive, uncompetitive, or noncompetitive
inhibition. An inhibitor can bind reversibly or irreversibly, and
therefore the term includes compounds that are suicide substrates
of an enzyme. An inhibitor can modify one or more sites on or near
the active site of the enzyme, or it can cause a conformational
change elsewhere on the enzyme.
[0064] The term "peptide" as used herein refers not only to
standard amide linkage with standard .alpha.-substituents, but also
to commonly used peptidomimetics, other modified linkages,
non-naturally occurring side chains, and side chain modifications,
for example, as described in U.S. Pat. No. 7,417,042.
[0065] The terms "polycyclyl" and "polycyclic" as used herein refer
to two or more rings (e.g., cycloalkyls, cycloalkenyls,
cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in which
two or more carbons are common to two adjoining rings, e.g., the
rings are "fused rings." Each of the rings of the polycycle can be
substituted or unsubstituted.
[0066] The term "prodrug" as used herein refers to compounds that,
under physiological conditions, are converted into therapeutically
active agents. A common method for making a prodrug is to include
selected moieties that are hydrolyzed under physiological
conditions to reveal the desired molecule. In other embodiments,
the prodrug is converted by an enzymatic activity of the host
animal.
[0067] The term "preventing" as used herein is art-recognized, and
when used in relation to a condition, such as a local recurrence
(e.g., pain), a disease such as cancer, a syndrome complex such as
heart failure or any other medical condition, is well understood in
the art, and includes administration of a composition that reduces
the frequency of, or delays the onset of, symptoms of a medical
condition in a subject relative to a subject who does not receive
the composition. Thus, prevention of cancer includes, for example,
reducing the number of detectable cancerous growths in a population
of subjects receiving a prophylactic treatment relative to an
untreated control population, and/or delaying the appearance of
detectable cancerous growths in a treated population versus an
untreated control population, e.g., by a statistically and/or
clinically significant amount. Prevention of an infection includes,
for example, reducing the number of diagnoses of the infection in a
treated population versus an untreated control population and/or
delaying the onset of symptoms of the infection in a treated
population versus an untreated control population. Prevention of
pain includes, for example, reducing the magnitude of, or
alternatively delaying, pain sensations experienced by subjects in
a treated population versus an untreated control population.
[0068] The term "cancer," as used herein, includes, but is not
limited to, blood borne and solid tumors.
[0069] The terms "autoimmune disease" and "autoimmune disorder," as
used herein refer to a disease or disorder arising from and
directed against an individual's own tissues.
[0070] The term "graft" as used herein refers to biological
material derived from a donor for transplantation into a
recipient.
[0071] The term "prophylactic or therapeutic" treatment, as used
herein, is art-recognized and refers to administration to the
subject of one or more of the subject compositions. If it is
administered prior to clinical manifestation of the unwanted
condition (e.g., disease or other unwanted state of the subject)
then the treatment is prophylactic, (i.e., it protects the subject
against developing the unwanted condition), whereas if it is
administered after manifestation of the unwanted condition, the
treatment is therapeutic, (i.e., it is intended to diminish,
ameliorate, or stabilize the existing unwanted condition or side
effects thereof).
[0072] The term "proteasome" as used herein refers to immuno- and
constitutive proteasomes.
[0073] The term "therapeutically effective amount" as used herein
refers to an amount of the compound(s) (e.g., a peptide epoxyketone
compound) in a preparation that, when administered as part of a
desired dosage regimen (to a mammal, preferably a human) alleviates
a symptom, ameliorates a condition, or slows the onset of disease
conditions according to clinically acceptable standards for the
disorder or condition to be treated or the cosmetic purpose, e.g.,
at a reasonable benefit/risk ratio applicable to any medical
treatment.
[0074] The terms "treating" and "treatment" as used herein refer to
reversing, reducing, or arresting the symptoms, clinical signs, and
underlying pathology of a condition in manner to improve or
stabilize a subject's condition.
[0075] The term "amphipathic lipids" as used herein refers to
molecules that are mostly lipid-like (hydrophobic) in structure,
but at one end have a region that is polar, charged, or a
combination of polar and charged (hydrophilic). The hydrophilic
region is referred to as the head group, and the lipid portion is
known as the tail group(s). Examples of amphipathic lipids include
phospholipids, glycolipids, and sphingolipids.
[0076] The terms "hydrophilic polymer" and "water-soluble polymer"
as used herein refer to polymers, for example, polyethylene glycol
(PEG) or other polyethoxylated polymers, used to shield liposomes
and thereby enhance liposomal circulatory half-life. "Hydrophilic
polymer" encompasses free hydrophilic polymers associated
non-covalently with the liposomes and hydrophilic polymers that are
conjugated or covalently linked to a component of the liposome
(e.g., PEG-derivatized lipids). Additional exemplary hydrophilic
polymers include, but are not limited to, polyvinyl alcohol,
polylactic acid, polyglycolic acid, polyacrylamide, polyglycerol,
poly(oxazoline), poly(N-(2-hydroxypropyl)methacrylamide)),
poly-N-vinylpyrrolidone, and poly(amino acid)s (PAA) (including,
for example, poly(hydroxyethyl
l-glutamine)-N-succinyldioctadecylamine (PHEG-DODASuc) and
poly(hydroxyl-ethyl l-asparagine)-DODASuc (PHEA-DODASuc)).
[0077] The term "free sterol" as used herein refers to a sterol
that is not covalently bound to another compound. "Free
cholesterol" refers to cholesterol that is not covalently bound as
a moiety in a sterol-modified amphiphilic lipid compound.
[0078] The terms "sterol" and "steroid alcohols" as used herein
refer to the subgroup of steroids having a free hydroxyl or a
derivative thereof. Exemplary sterols include, but are not limited
to, the class cholesterol and derivatives thereof, the class
phytosterols and derivatives thereof, and the class fungal sterols
and derivatives thereof. Sterols can be natural or synthetic.
[0079] The term "sterol-modified amphiphilic lipid" as used herein
refers to amphiphilic lipid compounds having a hydrophilic head
group, and two or more hydrophobic tails of which at least one is
sterol. "Sterol-modified amphiphilic phospholipids" refers to a
sterol-modified amphiphilic lipid comprising a phosphate-containing
moiety, such as phosphocholine or phosphoglycerol.
[0080] The term "therapeutic agent" as used herein refers to an
agent used in testing, development, or application as a
therapeutic, including drugs and pharmaceutical agents.
[0081] The term "drug" as used herein refers to any chemical
compound (e.g., a peptide epoxyketone compound) used in the
diagnosis, treatment, or prevention of disease or other abnormal
condition.
[0082] The term "prodrug" as used herein refers to compounds that,
under physiological conditions, are converted into therapeutically
active agents. A common method for making a prodrug is to include
selected moieties that are hydrolyzed under physiological
conditions to reveal the desired molecule. In other embodiments,
the prodrug is converted by an enzymatic activity of the
subject.
[0083] The terms "therapeutically acceptable" and "pharmaceutically
acceptable" as used herein refer to a material that is not
biologically or otherwise undesirable, i.e., the material can be
administered to a subject together with an active ingredient
without causing undesirable biological effects or interacting
adversely with any other component of the composition.
[0084] The term "emulsion" as used herein refers to a mixture of
two immiscible (unblendable) substances.
[0085] The term "bilayer" as used herein refers to a structure
composed of amphiphilic lipid molecules (often phospholipids)
arranged in two molecular layers, with the hydrophobic tails on the
interior and the polar head groups on the exterior surfaces.
[0086] The term "monolayer" as used herein refers to a single
molecular layer of amphipathic molecules with the head groups
aligned on one side, and hydrophobic groups on the opposite
side.
[0087] The term "liposome" as used herein refers to a vesicle
comprising a lipid bilayer, for example, a closed vesicle formed
when amphipathic lipids (e.g., phospholipids or their derivatives)
are dispersed in water. The liposomes of the present invention
typically comprise one or more phospholipids, and may also contain
mixed lipid chains with surfactant properties (e.g., egg
phosphatidylethanolamine). Liposome can employ surface ligands to
target binding to unhealthy tissue (e.g., tumors or neoplastic
cells). Liposomes typically have an aqueous core.
[0088] The term "entrapped" as used herein refers to the
non-covalent association of peptide epoxyketone compounds with a
liposome bilayer and/or the liposome's interior aqueous volume
(also called the liposome's aqueous core).
[0089] The terms "liposomal composition" and "liposome-containing
composition" are used interchangeably herein and refer to liposome
formulations or mixtures comprising lipids (e.g., phospholipids,
hydrophilic polymer-derivatized lipids, sterol components such as
cholesterols, and combinations thereof) and peptide epoxyketone
compounds, and such liposome mixtures or formulations can further
comprise additional excipients. A liposomal composition typically
comprises an aqueous solution comprising the liposomes.
Encapsulated aqueous solution is aqueous solution in the aqueous
core of the liposomes. Non-encapsulated aqueous solution is aqueous
solution in which the liposomes are dispersed.
[0090] The term "excipient" as used herein typically refers to any
pharmacologically inactive substance used for in the formulation or
administration of the liposomal compositions of the present
invention, for example, phospholipid, buffer, a carrier or vehicle
(such as diluents), and so on. Examples of excipients useful in the
practice of the present invention are described herein.
[0091] The term "pH adjusting agent" as used herein refers to any
agent used to modify the pH of an aqueous solution. pH is adjusted
by using acidifying (e.g., acids) and alkalizing agents (e.g.,
salts of acids or bases). Acidifying agents are used in a
formulation to lower the pH and alkalizing agents are used to
increase the pH. pH adjusting agents include buffering systems
(e.g., combinations of acids and bases). Pharmaceutical
compositions of the present invention can contain one or more of
these agents to achieve a desirable pH either for preparation
(i.e., in bulk solution) of the composition or upon reconstitution
for therapeutic administration.
[0092] The term "solublizing agent" as used herein refers to an
agent, typically a compound, pH adjusting agent, or cosolvent, that
increases the solubility of a peptide epoxyketone compound in an
aqueous solution.
[0093] The term "physiological conditions" as used herein refers to
conditions compatible with living cells, e.g., predominantly
aqueous conditions of a temperature, pH, salinity, etc.
[0094] The terms "therapeutic composition," "pharmaceutical
composition," "therapeutic preparation," and "pharmaceutical
preparation" are used interchangeably herein and encompass
liposomal compositions of the present invention suitable for
application or administration to a subject, typically a human. In
general such compositions are safe, sterile or asceptic, and
preferably free of contaminants that are capable of eliciting
undesirable responses in the subject (i.e., the compound(s)
comprising the composition are pharmaceutically acceptable).
Compositions can be formulated for application or administration to
a subject in need thereof by a number of different routes of
administration including oral (i.e., administered by mouth or
alimentary canal) or parenteral (e.g., buccal, rectal, transdermal,
transmucosal, subcutaneous, intravenous, intraperitoneal,
intradermal, intratracheal, intrathecal, pulmonary, and the
like).
[0095] The term "aseptic conditions" as used herein typically
refers to manufacturing or processing conditions wherein the
manufactured product is free from contamination with pathogens.
[0096] The term "subject" as used herein refers to any member of
the subphylum chordata, including, without limitation, humans and
other primates, including non-human primates such as rhesus
macaque, chimpanzees and other apes and monkey species; farm
animals such as cattle, sheep, pigs, goats and horses; domestic
mammals such as dogs and cats; laboratory animals including rodents
such as mice, rats and guinea pigs; birds, including domestic, wild
and game birds such as chickens, turkeys and other gallinaceous
birds, ducks, geese; and the like. The term does not denote a
particular age. Thus, adult, young, and newborn individuals are
intended to be covered.
2.0.0 GENERAL OVERVIEW OF THE INVENTION
[0097] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particular
types of liposomes, particular sources of drugs, particular lipids,
particular polymers, and the like, as use of such particulars can
be selected in view of the teachings of the present specification.
It is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments of the invention
only, and is not intended to be limiting.
[0098] Peptide epoxyketone compounds (e.g., carfilzomib) are
proteasome inhibitors useful for the treatment of a wide variety of
diseases and conditions. At present, carfilzomib for injection is
prepared by dissolving carfilzomib drug substance in
sulfobutylether beta cyclodextrin (SBE-B-CD) with citric acid using
a slurry method to create a bulk solution that is then lyophilized
to yield a lyophilized carfilzomib suitable for reconstitution and
injection.
[0099] However, intravenous administration of the carfilzomib
SBE-B-CD composition results in a short half-life due to rapid
metabolism. Clearance of carfilzomib is largely extrahepatic, and
carfilzomib is predominantly eliminated by peptidase cleavage and
epoxide hydrolysis. Therefore, multiple weekly injections are used
for treatment regimens. In addition, the use of the SBE-B-CD
composition can limit dose increases of carfilzomib, which can
impact its best profile activities.
[0100] Liposomes are spherical vesicles, typically comprising
phospholipids, that have an internal aqueous volume that is
enclosed by one or more concentric lipid bilayers with the polar
head groups oriented towards the interior and exterior aqueous
phases. Natural phospholipids are biocompatible and biodegradable
as they are naturally occurring in the body and are a major
constituent of cell membranes. Liposomes can act as drug carriers
by entrapping drugs in the aqueous core and/or within the lipid
bilayers. Liposomes range in size and can exist as unilamellar or
multilamellar vesicles.
[0101] The present application describes the successful development
of a variety of pharmaceutical liposomal compositions incorporating
peptide epoxyketone proteasome inhibitors. In some aspects of the
present invention, entrapment of peptide epoxyketone compounds are
described. In other aspects of the present invention, incorporation
of peptide epoxyketone compounds into the interior aqueous core of
liposomes are described. The liposomal compositions comprising
peptide epoxyketone compounds described herein enhance the
therapeutic window of peptide epoxyketone compounds by: improving
in vivo half-life (e.g., plasma half-life) relative to
non-liposomal compositions comprising peptide epoxyketone
compounds; providing desirable pharmacodynamic profiles (e.g.,
biodistribution, proteasome chymotrypsin-like (CT-L) activity
inhibition, and prolonged inhibition of proteasome CT-L activity in
selected tissues); and providing anti-tumor activity in a human
tumor xenograft model, greater than or equal to non-liposomal
compositions comprising peptide epoxyketone compounds. Further,
experiments performed in support of the present invention
demonstrated improved tolerability of liposomal compositions
comprising peptide epoxyketone compounds (e.g., improving maximum
tolerated dose relative to non-liposomal compositions comprising
peptide epoxyketone compounds).
[0102] The pharmaceutical liposomal compositions of the present
invention are typically prepared to be sterile or asceptic
compositions, and methods of making the pharmaceutical liposomal
compositions suitable for administration to a subject are typically
carried out under sterile or asceptic conditions. Terminal
sterilization of the pharmaceutical liposomal compositions of the
present invention can also be employed.
[0103] In a first aspect, the present invention relates to
pharmaceutical liposomal compositions. In some embodiments, the
pharmaceutical liposomal compositions comprise liposome entrapped
peptide epoxyketone compound. Such pharmaceutical compositions
typically comprise an aqueous solution comprising liposomes,
wherein the liposomes comprise between about 0.5 wt. % and about 50
wt. % of a peptide epoxyketone compound, and between about 99.5 wt.
% and about 50 wt. % total lipids (weight ratio of peptide
epoxyketone compound:total lipids of between about 0.005:0.995 and
about 0.5:0.5). In preferred embodiments the total lipids comprise
a phospholipid selected from the group consisting of
L-.alpha.-phosphatidylcholine;
1,2-distearoyl-sn-glycero-3-phosphocholine;
1,2-dipalmitoyl-sn-glycero-3-phosphocholine;
1,2-Distearoyl-sn-glycero-3-phospho-rac-(1-glycerol);
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; sphingomyelin;
1,2-distearoyl-sn-glycero-3-phosphoethanolamine; as well as
combinations thereof.
[0104] In embodiments of the pharmaceutical liposomal compositions
of the present invention, the weight ratio of peptide epoxyketone
compound:total lipid is between about 0.005:0.995 and about
0.35:0.65 (in weight percent, between about 0.5 wt. % and about 35
wt. % peptide epoxyketone compound and between about 99.5 wt. % and
about 65 wt. % total lipids), preferably between about 0.01:0.99
and about 0.30:0.70 (in weight percent, between about 1 wt. % and
about 30 wt. % peptide epoxyketone compound and between about 99
wt. % and about 70 wt. % total lipids), and more preferably between
about 0.01:0.99 and about 0.25:0.75 (in weight percent, between
about 1 wt. % and about 25 wt. % peptide epoxyketone compound and
between about 99 wt. % and about 75 wt. % total lipids).
[0105] In some embodiments of the pharmaceutical liposomal
compositions of the present invention, the total lipids of the
liposomes comprise between about 20 wt. % to about 100 wt. %
phospholipid. In preferred embodiments of the pharmaceutical
liposomal compositions of the present invention, the weight
percents of phospholipid include, but are not limited to, the
following: wherein the total lipids of the liposomes comprise
between about 30 wt. % and about 90 wt. % phospholipid, preferably
between about 50 wt. % and about 75 wt. % phospholipid.
[0106] In further embodiments, the total lipids of the liposomes
comprise a hydrophilic polymer-derivatized lipid, for example,
wherein the total lipids comprise between about 0.1 wt. % and about
30 wt. % of a hydrophilic polymer-derivatized lipid, between about
5 wt. % and about 25 wt. % of a hydrophilic polymer-derivatized
lipid, and preferably between about 8 wt. % and about 20 wt. % of a
hydrophilic polymer-derivatized lipid. Exemplary embodiments
include, but are not limited to, liposomes of the pharmaceutical
liposomal composition comprising between about 90 wt. % of the
phospholipid and about 75 wt. % of the phospholipid, and between
about 10 wt. % of the hydrophilic polymer-derivatized lipid and
about 25 wt. % of the hydrophilic polymer-derivatized lipid (total
lipid weight ratio of phospholipid:hydrophilic polymer-derivatized
lipid: {cholesterol or cholesterol derivative} of between about
0.9:0.1:0 and about 0.75:0.25:0). In embodiments wherein the total
lipids comprise a hydrophilic polymer-derivatized lipid, the lipid
of the hydrophilic polymer-derivatized lipid is, for example,
cholesterol or a phospholipid. In some embodiments, the hydrophilic
polymer of a hydrophilic polymer-derivatized lipid is a
polyethylene glycol. In a preferred embodiment, the hydrophilic
polymer-derivatized lipid is
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (mPEG2000DSPE).
[0107] In further embodiments, the total lipids of the liposomes
comprise a cholesterol or cholesterol derivative, wherein the total
lipids comprise between about 10 wt. % and about 50 wt. % a
cholesterol or cholesterol derivative, between about 15 wt. % and
about 40 wt. % a cholesterol or cholesterol derivative, preferably
between about 15 wt. % and about 30 wt. % a cholesterol or
cholesterol derivative. Exemplary embodiments include, but are not
limited to, liposomes of the pharmaceutical liposomal composition
comprising between about 90 wt. % and about 50 wt. % of the
phospholipid, and between about 10 wt. % and about 50 wt. % of a
cholesterol or derivative (total lipid weight ratio of
phospholipid:hydrophilic polymer-derivatized lipid: {cholesterol or
cholesterol derivative} of between about 0.9:0:0.1 and about
0.5:0:0.5). In preferred embodiments wherein the total lipids
comprise a cholesterol or cholesterol derivative, the cholesterol
or cholesterol derivative is cholesterol.
[0108] In additional embodiments, the total lipids of the liposomes
comprise a phospholipid, a hydrophilic polymer-derivatized lipid,
and a cholesterol or cholesterol derivative. Exemplary embodiments
include, but are not limited to, liposomes of the pharmaceutical
liposomal composition that comprise total lipids of between about
83.3 wt. % of the phospholipid and about 57 wt. % of the
phospholipid, between about 8.33 wt. % of the hydrophilic
polymer-derivatized lipid and about 14 wt. % of the hydrophilic
polymer-derivatized lipid, and between about 8.33 wt. % of the
cholesterol or cholesterol derivative and about 29 wt. % of the
cholesterol or cholesterol derivative (total lipid weight ratio of
phospholipid:hydrophilic polymer-derivatized lipid: {cholesterol or
cholesterol derivative} of between about 0.833:0.0833:0.0833 and
about 0.57:0.14:0.29).
[0109] The liposomes of the liposomal compositions of the present
invention typically have an average size of between about 0.05
microns and about 0.5 microns, between about 0.05 microns and about
0.2 microns, between about 0.05 microns and about 0.15 microns, and
preferably between about 0.05 microns and about 0.10 microns.
[0110] The lipids of the liposomes, in some embodiments, further
comprise .alpha.-tocopherol, for example, at about 0.001 to about 5
weight percent.
[0111] The aqueous solution in which the liposomes are dispersed
can also comprise one or more excipients, including, but not
limited to, a pH adjusting agent (e.g., a buffer) and/or an agent
to maintain isotonicity.
[0112] In other embodiments of this first aspect of the present
invention, the liposomal compositions comprise liposomes comprising
the peptide epoxyketone compound and a solubilizing agent in an
internal aqueous core of the liposomes. In some embodiments, the
solubilizing agent is a compound (e.g., a cyclodextrin), and the
liposomes of the liposomal composition comprise the peptide
epoxyketone compound complexed with the compound (e.g., a
cyclodextrin) in the internal aqueous core of the liposomes. A
preferred solubilizing agent that is a compound is a cyclodextrin,
for example, a sulfobutylether-betacyclodextrin or a
hydroxypropyl-betacyclodextrin.
[0113] The pharmaceutical liposomal composition of the present
invention can also include liposomal compositions wherein the
aqueous solution is adjusted to a pH of between about pH 3.0 and
about pH 7.0. Preferably, the aqueous solution is adjusted to a
human physiological pH.
[0114] Examples of peptide epoxyketone compounds for use in
liposomal compositions of the present invention include, but are
not limited to, compound I. Preferred peptide epoxyketone compounds
for use in liposomal compositions include compound II, compound
III, compound IV, and, most preferably carfilzomib (compound
V).
[0115] Preferred embodiments of pharmaceutical liposomal
compositions comprising peptide epoxyketone compounds include, but
are not limited to, the following: peptide epoxyketone
compound-EPC-mPEG2000DSPE-cholesterol; peptide epoxyketone
compound-sphingomyelin-mPEG2000DSPE-cholesterol; and peptide
epoxyketone compound-HSPC-mPEG2000DSPE-cholesterol.
[0116] In a second aspect, the present invention relates to dry
pharmaceutical compositions formed by drying the pharmaceutical
liposomal compositions described herein.
[0117] In a third aspect, the present invention relates to dry
pharmaceutical compositions comprising peptide epoxyketone
compounds. One embodiment of this third aspect of the present
invention is a dry pharmaceutical composition comprising between
about 0.5 wt. % and about 50 wt. % of a peptide epoxyketone
compound, and between about 99.5 wt. % and about 50 wt. % total
lipids (weight ratio of peptide epoxyketone compound:total lipids
of between about 0.005:0.995 and about 0.5:0.5). In preferred
embodiments, the total lipids comprise a phospholipid selected from
the group consisting of L-.alpha.-phosphatidylcholine;
1,2-distearoyl-sn-glycero-3-phosphocholine;
1,2-dipalmitoyl-sn-glycero-3-phosphocholine;
1,2-Distearoyl-sn-glycero-3-phospho-rac-(1-glycerol);
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; sphingomyelin;
1,2-distearoyl-sn-glycero-3-phosphoethanolamine; and combinations
thereof.
[0118] In embodiments of the dry pharmaceutical compositions of the
present invention, the weight ratio of peptide epoxyketone
compound:total lipid is between about 0.005:0.995 and about
0.35:0.65 (in weight percent, between about 0.5 wt. % and about 35
wt. % peptide epoxyketone compound and between about 99.5 wt. % and
about 65 wt. % total lipids), preferably between about 0.01:0.99
and about 0.30:0.70 (in weight percent, between about 1 wt. % and
about 30 wt. % peptide epoxyketone compound and between about 99
wt. % and about 70 wt. % total lipids), and more preferably between
about 0.01:0.99 and about 0.25:0.75 (in weight percent, between
about 1 wt. % and about 25 wt. % peptide epoxyketone compound and
between about 99 wt. % and about 75 wt. % total lipids).
[0119] In some embodiments of the dry pharmaceutical compositions
of the present invention, the total lipids comprise between about
20 wt. % to about 100 wt. % phospholipid. In preferred embodiments
of the dry pharmaceutical compositions of the present invention,
the weight percents of phospholipid include, but are not limited
to, the following: wherein the total lipids comprise between about
30 wt. % and about 90 wt. % phospholipid, preferably between about
50 wt. % and about 75 wt. % phospholipid.
[0120] In further embodiments, the total lipids of the dry
pharmaceutical compositions comprise a hydrophilic
polymer-derivatized lipid, for example, wherein the total lipids
comprise between about 0.1 wt. % and about 30 wt. % of a
hydrophilic polymer-derivatized lipid, between about 5 wt. % and
about 25 wt. % of a hydrophilic polymer-derivatized lipid, and
preferably between about 8 wt. % and about 20 wt. % of a
hydrophilic polymer-derivatized lipid. Exemplary embodiments
include, but are not limited to, dry pharmaceutical compositions
comprising between about 90 wt. % of the phospholipid and about 75
wt. % of the phospholipid, and between about 10 wt. % of the
hydrophilic polymer-derivatized lipid and about 25 wt. % of the
hydrophilic polymer-derivatized lipid (total lipid weight ratio of
phospholipid:hydrophilic polymer-derivatized lipid: {cholesterol or
cholesterol derivative} of between about 0.9:0.1:0 and about
0.75:0.25:0). In embodiments wherein the total lipids comprise a
hydrophilic polymer-derivatized lipid, the lipid of the hydrophilic
polymer-derivatized lipid is, for example, cholesterol or a
phospholipid. In some embodiments, the hydrophilic polymer of a
hydrophilic polymer-derivatized lipid is a polyethylene glycol. In
a preferred embodiment, the hydrophilic polymer-derivatized lipid
is
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (mPEG2000DSPE).
[0121] In further embodiments, the total lipids of the dry
pharmaceutical compositions comprise a cholesterol or cholesterol
derivative, wherein the total lipids comprise between about 10 wt.
% and about 50 wt. % a cholesterol or cholesterol derivative,
between about 15 wt. % and about 40 wt. % a cholesterol or
cholesterol derivative, preferably between about 15 wt. % and about
30 wt. % a cholesterol or cholesterol derivative. Exemplary
embodiments include, but are not limited to, dry pharmaceutical
compositions comprising between about 90 wt. % and about 50 wt. %
of the phospholipid, and between about 10 wt. % and about 50 wt. %
of a cholesterol or derivative (total lipid weight ratio of
phospholipid:hydrophilic polymer-derivatized lipid: {cholesterol or
cholesterol derivative} of between about 0.9:0:0.1 and about
0.5:0:0.5). In preferred embodiments wherein the total lipids
comprise a cholesterol or cholesterol derivative, the cholesterol
or cholesterol derivative is cholesterol.
[0122] In additional embodiments, the total lipids of the dry
pharmaceutical compositions comprise a phospholipid, a hydrophilic
polymer-derivatized lipid, and a cholesterol or cholesterol
derivative. Exemplary embodiments include, but are not limited to,
dry pharmaceutical compositions that comprise total lipids of
between about 83.3 wt. % of the phospholipid and about 57 wt. % of
the phospholipid, between about 8.33 wt. % of the hydrophilic
polymer-derivatized lipid and about 14 wt. % of the hydrophilic
polymer-derivatized lipid, and between about 8.33 wt. % of the
cholesterol or cholesterol derivative and about 29 wt. % of the
cholesterol or cholesterol derivative (total lipid weight ratio of
phospholipid:hydrophilic polymer-derivatized lipid: {cholesterol or
cholesterol derivative} of between about 0.833:0.0833:0.0833 and
about 0.57:0.14:0.29).
[0123] The dry pharmaceutical compositions, in some embodiments,
further comprise .alpha.-tocopherol, for example, at about 0.001 to
about 5 weight percent.
[0124] The dry pharmaceutical compositions comprising peptide
epoxyketone compounds can also comprise one or more excipients,
including, but not limited to, a pH adjusting agent (e.g., a
buffer) and/or an agent to maintain isotonicity.
[0125] In other embodiments of this third aspect of the present
invention, the dry pharmaceutical compositions comprise the peptide
epoxyketone compound and a cyclodextrin. Preferred cyclodextrins
include sulfobutylether-betacyclodextrins or
hydroxypropyl-betacyclodextrins.
[0126] Examples of peptide epoxyketone compounds for use in dry
pharmaceutical compositions of the present invention include, but
are not limited to, compound I. Preferred peptide epoxyketone
compounds for use in dry pharmaceutical compositions include
compound II, compound III, compound IV, and, most preferably
carfilzomib (compound V).
[0127] In some embodiments, dry pharmaceutical compositions further
comprise additional excipients, for example cryoprotectant agents
(e.g., glycerol, dimethylamine, dimethylsulfoxide), glass
transition modifying agents (e.g. sugars, polyols, polymers, amino
acids), combinations thereof, and/or other stabilizing
excipients.
[0128] Preferred embodiments of dry pharmaceutical compositions
comprising peptide epoxyketone compounds include, but are not
limited to, the following: peptide epoxyketone
compound-EPC-mPEG2000DSPE-cholesterol; peptide epoxyketone
compound-sphingomyelin-mPEG2000DSPE-cholesterol; and peptide
epoxyketone compound-HSPC-mPEG2000DSPE-cholesterol.
[0129] In a fourth aspect, the present invention relates to a
method of making pharmaceutical liposomal compositions comprising
reconstituting dry pharmaceutical compositions comprising peptide
epoxyketone compounds and lipids (for example, as described in the
third aspect of the present invention) using an aqueous solution to
form liposomes, the pharmaceutical liposomal composition comprising
the aqueous solution comprising the liposomes.
[0130] In a fifth aspect, the present invention relates to
pharmaceutical liposomal compositions made by the method described
in the fourth aspect of the present invention. The liposomes of the
pharmaceutical liposomal compositions have, for example, liposomes
with an average size of between about 0.05 microns and about 0.5
microns, between about 0.05 microns and about 0.2 microns, between
about 0.05 microns and about 0.15 microns, and preferably between
about 0.05 microns and about 0.10 microns.
[0131] In a sixth aspect, the present invention relates to a method
of making a pharmaceutical liposomal composition comprising
preparing a dried film comprising total lipids, and rehydrating the
dried film with an aqueous solution comprising a peptide
epoxyketone compound and a solubilizing agent to form the
pharmaceutical liposomal composition. Typically the method
comprises preparing a dried film comprising total lipids, wherein
the total lipids comprise a phospholipid selected from the group
consisting of L-.alpha.-phosphatidylcholine;
1,2-distearoyl-sn-glycero-3-phosphocholine;
1,2-dipalmitoyl-sn-glycero-3-phosphocholine;
1,2-Distearoyl-sn-glycero-3-phospho-rac-(1-glycerol);
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; sphingomyelin;
1,2-distearoyl-sn-glycero-3-phosphoethanolamine; and a combination
thereof. The dried film is rehydrated with an aqueous solution
comprising a peptide epoxyketone compound and a solubilizing agent
to form the pharmaceutical liposomal composition comprising
liposomes dispersed in the aqueous solution; wherein the liposomes
comprise (i) between about 0.5 wt. % and about 50 wt. % of the
peptide epoxyketone compound entrapped in the liposomes, and (ii)
between about 99.5 wt. % and about 50 wt. % of the total lipids
(i.e., weight ratio of peptide epoxyketone compound:total lipids of
between about 0.005:0.995 and about 0.5:0.5).
[0132] In embodiments of the methods of this sixth aspect of the
present invention, the weight ratio of peptide epoxyketone
compound:total lipid is between about 0.005:0.995 and about
0.35:0.65 (in weight percent, between about 0.5 wt. % and about 35
wt. % peptide epoxyketone compound and between about 99.5 wt. % and
about 65 wt. % total lipids), preferably between about 0.01:0.99
and about 0.30:0.70 (in weight percent, between about 1 wt. % and
about 30 wt. % peptide epoxyketone compound and between about 99
wt. % and about 70 wt. % total lipids), and more preferably between
about 0.01:0.99 and about 0.25:0.75 (in weight percent, between
about 1 wt. % and about 25 wt. % peptide epoxyketone compound and
between about 99 wt. % and about 75 wt. % total lipids).
[0133] In some embodiments of the methods of this sixth aspect of
the present invention, the total lipids comprise between about 20
wt. % to about 100 wt. % phospholipid. In preferred embodiments of
the method, the weight percents of phospholipid include, but are
not limited to, the following: wherein the total lipids comprise
between about 30 wt. % and about 90 wt. % phospholipid, preferably
between about 50 wt. % and about 75 wt. % phospholipid.
[0134] In further embodiments of the method, the total lipids
comprise a hydrophilic polymer-derivatized lipid, for example,
wherein the total lipids comprise between about 0.1 wt. % and about
30 wt. % of a hydrophilic polymer-derivatized lipid, between about
5 wt. % and about 25 wt. % of a hydrophilic polymer-derivatized
lipid, and preferably between about 8 wt. % and about 20 wt. % of a
hydrophilic polymer-derivatized lipid. Exemplary embodiments
include, but are not limited to, wherein the total lipids comprise
between about 90 wt. % of the phospholipid and about 75 wt. % of
the phospholipid, and between about 10 wt. % of the hydrophilic
polymer-derivatized lipid and about 25 wt. % of the hydrophilic
polymer-derivatized lipid (total lipid weight ratio of
phospholipid:hydrophilic polymer-derivatized lipid: {cholesterol or
cholesterol derivative} of between about 0.9:0.1:0 and about
0.75:0.25:0). In embodiments wherein the total lipids comprise a
hydrophilic polymer-derivatized lipid, the lipid of the hydrophilic
polymer-derivatized lipid is, for example, cholesterol or a
phospholipid. In some embodiments, the hydrophilic polymer of a
hydrophilic polymer-derivatized lipid is a polyethylene glycol. In
a preferred embodiment, the hydrophilic polymer-derivatized lipid
is
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (mPEG2000DSPE).
[0135] In further embodiments of the methods of this sixth aspect
of the present invention, the total lipids comprise a cholesterol
or cholesterol derivative, wherein the total lipids comprise
between about 10 wt. % and about 50 wt. % a cholesterol or
cholesterol derivative, between about 15 wt. % and about 40 wt. % a
cholesterol or cholesterol derivative, preferably between about 15
wt. % and about 30 wt. % a cholesterol or cholesterol derivative.
Exemplary embodiments include, but are not limited to, total lipids
comprising between about 90 wt. % and about 50 wt. % of the
phospholipid, and between about 10 wt. % and about 50 wt. % of a
cholesterol or derivative (total lipid weight ratio of
phospholipid:hydrophilic polymer-derivatized lipid: {cholesterol or
cholesterol derivative} of between about 0.9:0:0.1 and about
0.5:0:0.5). In preferred embodiments wherein the total lipids
comprise a cholesterol or cholesterol derivative, the cholesterol
or cholesterol derivative is cholesterol.
[0136] In additional embodiments of the method, the total lipids
comprise a phospholipid, a hydrophilic polymer-derivatized lipid,
and a cholesterol or cholesterol derivative. Exemplary embodiments
include, but are not limited to, total lipids of between about 83.3
wt. % of the phospholipid and about 57 wt. % of the phospholipid,
between about 8.33 wt. % of the hydrophilic polymer-derivatized
lipid and about 14 wt. % of the hydrophilic polymer-derivatized
lipid, and between about 8.33 wt. % of the cholesterol or
cholesterol derivative and about 29 wt. % of the cholesterol or
cholesterol derivative (total lipid weight ratio of
phospholipid:hydrophilic polymer-derivatized lipid: {cholesterol or
cholesterol derivative} of between about 0.833:0.0833:0.0833 and
about 0.57:0.14:0.29).
[0137] In some embodiments of the method of this sixth aspect of
the present invention the solubilizing agent is, for example, a
compound, a pH adjusting agent, a cosolvent, or a combination
thereof. In some embodiments, the solubilizing agent is a compound
(e.g., a cyclodextrin), and the liposomes of the liposomal
composition comprise the peptide epoxyketone compound complexed
with the compound (e.g., a cyclodextrin) in the internal aqueous
core of the liposomes. A preferred solubilizing agent that is a
compound is a cyclodextrin, for example, a
sulfobutylether-betacyclodextrin or a
hydroxypropyl-betacyclodextrin. In other embodiments, the
solubilizing agent comprises a pH adjusting agent and the aqueous
solution has a pH of between about pH 0.5 and about pH 3, between
about pH 0.5 and about pH 2, and preferably between about pH 1 and
about pH 2. In further embodiments, the solubilizing agent
comprises a cosolvent.
[0138] The methods of this sixth aspect of the present invention
can further include dialysis, desalting, buffer exchange, and/or
gel filtration.
[0139] The method can further comprise sizing the liposomes to have
an average size of between about 0.05 microns and about 0.5
microns, an average size of between about 0.05 microns and about
0.2 microns, between about 0.05 microns and about 0.15 microns, and
preferably between about 0.05 microns and about 0.10 microns.
[0140] In some embodiments, the method further comprises, after
forming the liposomal composition (wherein the liposomal
composition comprises aqueous solution encapsulated in the
liposomes and aqueous solution not encapsulated in the liposomes,
i.e., non-encapsulated aqueous solution), removing peptide
epoxyketone compound from the non-encapsulated aqueous solution in
which the liposomes are dispersed. Removal of peptide expoxyketone
compounds from the non-encapsulated aqueous solution can be
accomplished, for example, using dialysis, ultracentrifugation, gel
filtration, or combinations thereof.
[0141] In some embodiments, the method further comprise, after
rehydrating the dried film to form the liposomal composition,
adjusting the pH of the aqueous solution. The pH can be adjusted
to, for example, a pH of between about pH 3.0 and about pH 7.0,
preferably to a human physiological pH.
[0142] The method can further comprise, after rehydrating the dried
film to form the liposomal composition, adding one or more
excipients to the aqueous solution, for example, a pH adjusting
agent (e.g., a buffer) and/or an agent to maintain isotonicity.
[0143] Examples of peptide epoxyketone compounds for use in the
method include, but are not limited to, compound I. Preferred
peptide epoxyketone compounds for use in liposomal compositions
include compound II, compound III, compound IV, and, most
preferably carfilzomib (compound V).
[0144] In a seventh aspect, the present invention relates to
pharmaceutical liposomal compositions made by the method of the
sixth aspect of the present invention; the liposomal composition
comprising liposomes dispersed in the aqueous solution, wherein the
liposomes comprise a peptide epoxyketone compound entrapped in the
liposomes.
[0145] In an eight aspect, the present invention relates to a dry
pharmaceutical composition formed by drying the pharmaceutical
liposomal composition of the seventh aspect of the invention.
[0146] In a ninth aspect, the present invention relates to a method
of making a pharmaceutical liposomal composition comprising
preparing a lipid solution and injecting the lipid solution into an
aqueous solution comprising a peptide epoxyketone compound. The
lipid solution comprises a solvent and total lipids. The total
lipids typically comprise a phospholipid, for example,
L-.alpha.-phosphatidylcholine,
1,2-distearoyl-sn-glycero-3-phosphocholine,
1,2-dipalmitoyl-sn-glycero-3-phosphocholine,
1,2-Distearoyl-sn-glycero-3-phospho-rac-(1-glycerol),
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, sphingomyelin,
1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and combinations
thereof. Typically the method comprises injecting the lipid
solution into an aqueous solution comprising a peptide epoxyketone
compound and a solubilizing agent to form the pharmaceutical
liposomal composition. The pharmaceutical liposomal composition
comprises liposomes dispersed in the aqueous solution, wherein the
liposomes comprise (i) between about 0.5 wt. % and about 50 wt. %
of the peptide epoxyketone compound entrapped in the liposomes, and
(ii) between about 99.5 wt. % and about 50 wt. % of the total
lipids (weight ratio of peptide epoxyketone compound:total lipids
of between about 0.005:0.995 and about 0.5:0.5).
[0147] In some embodiments the solvent is an organic solvent, for
example an alcohol (e.g., ethanol).
[0148] In embodiments of the methods of this ninth aspect of the
present invention, the weight ratio of peptide epoxyketone
compound:total lipid is between about 0.005:0.995 and about
0.35:0.65 (in weight percent, between about 0.5 wt. % and about 35
wt. % peptide epoxyketone compound and between about 99.5 wt. % and
about 65 wt. % total lipids), preferably between about 0.01:0.99
and about 0.30:0.70 (in weight percent, between about 1 wt. % and
about 30 wt. % peptide epoxyketone compound and between about 99
wt. % and about 70 wt. % total lipids), and more preferably between
about 0.01:0.99 and about 0.25:0.75 (in weight percent, between
about 1 wt. % and about 25 wt. % peptide epoxyketone compound and
between about 99 wt. % and about 75 wt. % total lipids).
[0149] In some embodiments of the methods of this ninth aspect of
the present invention, the total lipids comprise between about 20
wt. % to about 100 wt. % phospholipid. In preferred embodiments of
the method, the weight percents of phospholipid include, but are
not limited to, the following: wherein the total lipids comprise
between about 30 wt. % and about 90 wt. % phospholipid, preferably
between about 50 wt. % and about 75 wt. % phospholipid.
[0150] In further embodiments of the method, the total lipids
comprise a hydrophilic polymer-derivatized lipid, for example,
wherein the total lipids comprise between about 0.1 wt. % and about
30 wt. % of a hydrophilic polymer-derivatized lipid, between about
5 wt. % and about 25 wt. % of a hydrophilic polymer-derivatized
lipid, and preferably between about 8 wt. % and about 20 wt. % of a
hydrophilic polymer-derivatized lipid. Exemplary embodiments
include, but are not limited to, wherein the total lipids comprise
between about 90 wt. % of the phospholipid and about 75 wt. % of
the phospholipid, and between about 10 wt. % of the hydrophilic
polymer-derivatized lipid and about 25 wt. % of the hydrophilic
polymer-derivatized lipid (total lipid weight ratio of
phospholipid:hydrophilic polymer-derivatized lipid: {cholesterol or
cholesterol derivative} of between about 0.9:0.1:0 and about
0.75:0.25:0). In embodiments wherein the total lipids comprise a
hydrophilic polymer-derivatized lipid, the lipid of the hydrophilic
polymer-derivatized lipid is, for example, cholesterol or a
phospholipid. In some embodiments, the hydrophilic polymer of a
hydrophilic polymer-derivatized lipid is a polyethylene glycol. In
a preferred embodiment, the hydrophilic polymer-derivatized lipid
is
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (mPEG2000DSPE).
[0151] In further embodiments of the methods of this ninth aspect
of the present invention, the total lipids comprise a cholesterol
or cholesterol derivative, wherein the total lipids comprise
between about 10 wt. % and about 50 wt. % a cholesterol or
cholesterol derivative, between about 15 wt. % and about 40 wt. % a
cholesterol or cholesterol derivative, preferably between about 15
wt. % and about 30 wt. % a cholesterol or cholesterol derivative.
Exemplary embodiments include, but are not limited to, total lipids
comprising between about 90 wt. % and about 50 wt. % of the
phospholipid, and between about 10 wt. % and about 50 wt. % of a
cholesterol or derivative (total lipid weight ratio of
phospholipid:hydrophilic polymer-derivatized lipid: {cholesterol or
cholesterol derivative} of between about 0.9:0:0.1 and about
0.5:0:0.5). In preferred embodiments wherein the total lipids
comprise a cholesterol or cholesterol derivative, the cholesterol
or cholesterol derivative is cholesterol.
[0152] In additional embodiments of the method, the total lipids
comprise a phospholipid, a hydrophilic polymer-derivatized lipid,
and a cholesterol or cholesterol derivative. Exemplary embodiments
include, but are not limited to, total lipids of between about 83.3
wt. % of the phospholipid and about 57 wt. % of the phospholipid,
between about 8.33 wt. % of the hydrophilic polymer-derivatized
lipid and about 14 wt. % of the hydrophilic polymer-derivatized
lipid, and between about 8.33 wt. % of the cholesterol or
cholesterol derivative and about 29 wt. % of the cholesterol or
cholesterol derivative (total lipid weight ratio of
phospholipid:hydrophilic polymer-derivatized lipid: {cholesterol or
cholesterol derivative} of between about 0.833:0.0833:0.0833 and
about 0.57:0.14:0.29).
[0153] In some embodiments of the method of this ninth aspect of
the present invention the solubilizing agent is, for example, a
compound, a pH adjusting agent, a cosolvent, or a combination
thereof. In some embodiments, the solubilizing agent is a compound
(e.g., a cyclodextrin), and the liposomes of the liposomal
composition comprise the peptide epoxyketone compound complexed
with the compound (e.g., a cyclodextrin) in the internal aqueous
core of the liposomes. A preferred solubilizing agent that is a
compound is a cyclodextrin, for example, a
sulfobutylether-betacyclodextrin or a
hydroxypropyl-betacyclodextrin. In other embodiments, the
solubilizing agent comprises a pH adjusting agent and the aqueous
solution has a pH of between about pH 0.5 and about pH 3, between
about pH 0.5 and about pH 2, and preferably between about pH 1 and
about pH 2. In further embodiments, the solubilizing agent
comprises a cosolvent.
[0154] The method of this ninth aspect of the present invention can
further include dialysis, desalting, buffer exchange, and/or gel
filtration.
[0155] The method can further comprise sizing the liposomes to have
an average size of between about 0.05 microns and about 0.5
microns, an average size of between about 0.05 microns and about
0.2 microns, between about 0.05 microns and about 0.15 microns, and
preferably between about 0.05 microns and about 0.10 microns.
[0156] In some embodiments, the method further comprises, after
forming the liposomal composition (wherein the liposomal
composition comprises aqueous solution encapsulated in the
liposomes and aqueous solution not encapsulated in the liposomes,
i.e., non-encapsulated aqueous solution), removing peptide
epoxyketone compound from the non-encapsulated aqueous solution in
which the liposomes are dispersed. Removal of peptide expoxyketone
compounds from the non-encapsulated aqueous solution can be
accomplished, for example, using dialysis, ultracentrifugation, gel
filtration, or combinations thereof.
[0157] In some embodiments, the method further comprise, after
injecting the lipid solution into the aqueous solution to form the
liposomal composition, adjusting the pH of the aqueous solution.
The pH can be adjusted to, for example, a pH of between about pH
3.0 and about pH 7.0, preferably to a human physiological pH.
[0158] The method can further comprise, after injecting the lipid
solution into the aqueous solution to form the liposomal
composition, adding one or more excipients to the aqueous solution,
for example, a pH adjusting agent (e.g., a buffer) and/or an agent
to maintain isotonicity.
[0159] Examples of peptide epoxyketone compounds for use in the
method include, but are not limited to, compound I. Preferred
peptide epoxyketone compounds for use in liposomal compositions
include compound II, compound III, compound IV, and, most
preferably carfilzomib (compound V).
[0160] In a tenth aspect, the present invention relates to
pharmaceutical liposomal compositions made by the method of the
ninth aspect of the present invention; the liposomal composition
comprising liposomes dispersed in the aqueous solution, wherein the
liposomes comprise a peptide epoxyketone compound entrapped in the
liposomes.
[0161] In an eleventh aspect, the present invention relates to a
dry pharmaceutical composition formed by drying the pharmaceutical
liposomal composition of the tenth aspect of the invention.
[0162] In a twelfth aspect, the present invention relates to
methods of treating a disease or condition in a subject in need of
treatment, comprising administering a therapeutically effective
amount of a pharmaceutical liposomal composition, as described
herein, comprising liposomes comprising a peptide epoxyketone
compound. In some embodiments the methods of treating further
comprise simultaneous, sequential, or separate administration of a
therapeutically effective amount of another therapeutic agent, for
example, a chemotherapeutic agent, a cytokine, a steroid, an
immunotherapeutic agent, or combinations thereof. Examples of
diseases or conditions that are treated using the pharmaceutical
liposomal compositions of the present invention comprising peptide
epoxyketone compounds include, but are not limited to, multiple
myeloma, solid tumors, infections, and autoimmune diseases.
3.0.0 PHARMACEUTICAL COMPOSITIONS
[0163] The present invention relates to pharmaceutical liposomal
compositions comprising peptide epoxyketone compounds (e.g.,
carfilzomib) and prodrugs thereof, dry pharmaceutical compositions
comprising peptide epoxyketone compounds (e.g., carfilzomib) and
prodrugs thereof, and methods of making and using such
compositions.
[0164] 3.1.0 Peptide Epoxyketone Compounds
[0165] Examples of peptide epoxyketone compounds useful in the
practice of the present invention are described in U.S. Pat. No.
7,417,042, and include, but are not limited to, a peptide
epoxyketone compound having the structure of formula I:
##STR00004##
[0166] wherein X is O, NH, or N-alkyl; Y is NH, N-alkyl, O, or
C(R.sup.9).sub.2; Z is O or C(R.sup.9).sub.2; R.sup.1, R.sup.2,
R.sup.3, and R.sup.4 are all hydrogen; each R.sup.5, R.sup.6,
R.sup.7, R.sup.8, and R.sup.9 is independently selected from
hydrogen, C.sub.1-6alkyl, C.sub.1-6hydroxyalkyl,
C.sub.1-6alkoxyalkyl, aryl, and C.sub.1-6aralkyl, each of which is
optionally substituted with a group selected from alkyl, amide,
amine, carboxylic acid or a pharmaceutically acceptable salt
thereof, carboxyl ester, thiol, and thioether; m is an integer from
0 to 2; and n is an integer from 0 to 2. Terms used to describe
these compounds are further set forth in the "Definitions"
section.
[0167] Examples of specific peptide epoxyketone compounds useful in
the practice of the present invention include the following
compounds having formulas II, III, and IV ("Ph" in the following
compounds represents a phenyl group):
##STR00005##
[0168] In a preferred embodiment of the present invention, the
peptide epoxyketone compound is carfilzomib having formula V:
##STR00006##
[0169] In the liposomal compositions of the present invention, the
weight ratio of peptide epoxyketone compound:total lipid (wt.
drug:wt. total lipid) is typically between about 0.005:0.995 and
about 0.5:0.5 (in weight percent (wt. %) between about 0.5 wt. %
and about 50 wt. % drug and between about 99.5 wt. % and about 50
wt. % total lipids). In preferred embodiments, the weight ratio of
peptide epoxyketone compound:total lipid is between about
0.005:0.995 and about 0.35:0.65 (in weight percent, between about
0.5 wt. % and about 35 wt. % drug and between about 99.5 wt. % and
about 65 wt. % total lipids), preferably between about 0.01:0.99
and about 0.30:0.70 (in weight percent, between about 1 wt. % and
about 30 wt. % drug and between about 99 wt. % and about 70 wt. %
total lipids), and more preferably between about 0.01:0.99 and
about 0.25:0.75 (in weight percent, between about 1 wt. % and about
25 wt. % drug and between about 99 wt. % and about 75 wt. % total
lipids).
[0170] 3.2.0 Liposome Components
[0171] Types of lipids used in the practice of the present
invention include, but are not limited to phospholipids, sterols,
and modifications and derivatives thereof. Additional amphipathic
lipids can also be used in the practice of the present
invention.
[0172] Preferred vesicle forming amphipathic lipids for use in the
practice of the present invention include phospholipids and
derivatives thereof. Phospholipids fall generally into three
classes, neutral, cationic, and anionic.
[0173] Examples of phospholipids useful in the practice of the
present invention include, but are not limited to, the following:
phosphatidylcholine; L-.alpha.-phosphatidylcholine (egg
phosphatidylcholine (EPC), or hydrogenated soy phosphatidylcholine
(HSPC)); 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC);
phosphatidylserine (PS); phosphatidylinositol (PI);
phosphatidylglycerol (PG); phosphatidylethanolamine (PE); dioleoyl
phosphatidylglycerol (DOPG);
1,2-Dioleoyl-sn-glycero-3-phosphocholine (or dioleoyl
phosphatidylcholine) (DOPC); dioleoyl phosphatidylserine (DOPS);
1,2-dileoyl-sn-glycero-3-phosphoethanolamine (DOPE);
1,2-Dioleoyl-sn-glycero-3-phosphate (DOPA);
1-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC);
1,2-Dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DPPG);
1,2-Dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DMPG);
1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC);
1,2-Distearoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DSPG);
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC);
diacylphosphatidylcholine; diacylphosphatidic acid; N-dodecanoyl
phosphatidylethanolamine: N-succinyl phosphatidylethanolamine:
N-glutaryl phosphatidylethanolamine: lysylphosphatidylglycerol;
sphingolipids (e.g., sphingomyelin); and mixtures thereof.
[0174] Further vesicle forming lipids useful in the practice of the
present invention include, for example,
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC);
N-(2,3-dioleyloxyl)propyl-N,N--N-triethylammonium chloride (DOTMA);
N,N-distearyl-N,N-dimethylammonium bromide (DDAB);
N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP);
N-(1-(2,3-dioleyloxyl)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethy-
lammonium trifluoracetate (DOSPA);
dioctadecylamidoglycylcarboxyspermine (DOGS);
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide (DMRIE); stearylamine; dicetyl phosphate;
0-oleoyl-.gamma.-palmitoyl; and mixtures thereof.
[0175] Preferred lipids for use in the practice of the present
invention include, but are not limited to:
L-.alpha.-phosphatidylcholine (e.g., egg phosphatidylcholine (EPC),
or hydrogenated soy phosphatidylcholine (HSPC));
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC);
1,2-Distearoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DSPG);
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC);
sphingomyelin (SPH);
1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE); and
combinations thereof. In some embodiments, the lipids of the
liposomes comprise between about 20 to about 100 weight percent
phospholipid, between about 30 and about 90 weight percent
phospholipid, preferably between about 50 and about 75 weight
percent phospholipid.
[0176] A variety of sterols and derivatives thereof (e.g.,
cholesterol) can be used to stabilize liposomes. Sterol-modified
amphiphilic lipids are known in the art (see, e.g., U.S. Patent
Application Publication No. 2011/0177156). Sterols for use in the
practice of the present invention, such as cholesterol, also can be
derivatized with a variety of hydrophilic polymers (PEG-cholesterol
derivatives; see, e.g., U.S. Pat. No. 6,270,806). In an embodiment
of the present invention, sterols or derivatives thereof can be
added to the liposomal composition to stabilize the lipid bilayer.
Preferred sterols for use in the practice of the present invention
are cholesterol and its derivatives (e.g., cholesterol
hemisuccinates; hydroxycholesterols; cholestens; ketocholestanols;
cholesteryl acetates; cholesteryl linoleates; cholesteryl
dodecanoates; cholesteryl palmitates; thiocholesterols;
lysine-based cholesterols; hydroxyethylated cationic cholesterols).
For example, the lipids of the liposomes of the liposomal
compositions of the present invention can comprise between about 10
and about 50 weight percent cholesterol, between about 15 and about
40 weight percent cholesterol, preferably between about 15 and
about 30 weight percent cholesterol.
[0177] In other embodiments, cholesterol is chemically modified
with a ligand designed to be recognized by a particular organ or
cell type such as a long chain fatty acid, an amino acid, an
oligosaccharide, a hormone, an amino acid derivative, a protein,
glycoprotein, modified protein, or the like. The resultant liposome
is suitable for being targeted to a specific organ or cell type
(see, e.g., U.S. Pat. No. 4,544,545).
[0178] Additional examples of liposomal compositions including
targeting factors that can be used, in view of the teachings of the
present specification, include U.S. Pat. Nos. 5,049,390; 5,780,052;
5,786,214; 5,830,686; 6,056,973; 6,110,666; 6,177,059; 6,245,427;
6,316,024; 6,524,613; 6,530,944; 6,749,863; 6,803,360; 6,960,560;
7,060,291; 7,101,985; and U.S. Patent Application Nos.
2002/0198164; 2003/0027779; 2003/0220284; 2003/0224037;
2003/0228285; 2003/143742; and 2004/0022842.
[0179] Steric stabilization refers to the colloidal stability
conferred on the liposome by a variety of hydrophilic polymers or
hydrophilic glycolipids, for example, polyethylene glycol and the
ganglioside GM1. Liposomes can contain PEG-PE, GM1, or another such
glycolipid or polymer that demonstrates a relatively long half-life
in the general circulation. Hydrophilic polymers such as PEG and
other polyethoxylated polymers can be used to shield liposomes to
enhance the circulatory half-life of the liposome. Such hydrophilic
polymers can be associated non-covalently with the liposomes or
conjugated or covalently linked to a particular component of the
liposome (e.g., PEG-derivatized lipids; such as
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (ammonium salt) (mPEG-DSPE)). Additional exemplary
hydrophilic polymers include, but are not limited to, polyvinyl
alcohols, polylactic acids, polyglycolic acids,
polyvinylpyrrolidones, polyacrylamides, polyglycerols,
polyaxozlines, polyaminoacids (PAAs), and mixtures thereof.
[0180] In some embodiments of the liposomal compositions described
herein, the lipids of the liposomes can comprise between about 0.1
and about 30 weight percent of a hydrophilic polymer-derivatized
lipid, between about 5 and about 25 weight percent of a hydrophilic
polymer-derivatized lipid, preferably between about 8 and about 20
weight percent of a hydrophilic polymer-derivatized lipid.
Preferred hydrophilic polymers for use in the practice of the
present invention are polyethylene glycols (e.g., phospholipids
conjugated to monomethoxy polyethylene glycol, for example,
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (mPEG-DSPE)).
[0181] Additional examples of liposomal compositions that can be
used, in view of the teachings of the present specification,
include: U.S. Pat. Nos. 4,789,633; 4,925,661; 4,983,397; 5,013,556;
5,534,241; 5,593,622; 5,648,478; 5,676,971; 5,756,069; 5,834,012;
5,846,458; 5,891,468; 5,945,122; 6,056,973; 6,057,299; 6,077,834;
6,126,966; 6,153,596; 6,287,593; 6,316,024; 6,387,397; 6,476,068;
6,586,559; 6,627,218; 6,723,338; 6,897,196; 6,936,272; 6,960,560;
7,122,202; 7,311,924; 7,361,640; and 7,901,708; and U.S. Patent
Application Publication Nos. 2003/0072794; 2003/0082228;
2003/0166601; 2003/0203865 2003/0215490; 2003/0224037;
2004/0022842; 2004/0234588; and 2005/0136064.
[0182] The liposomal compositions typically comprise liposome
entrapped peptide epoxyketone compounds and an aqueous carrier.
[0183] Typical excipients useful in the practice of the present
invention include, but are not limited to, the following: carrier
or vehicle (e.g., water or buffered aqueous solutions); pH
adjusting agents; antioxidants (e.g., .alpha.-tocopherol,
methionine, ascorbic acid, sodium thiosulfate,
ethylenediaminetetraacetic acid, citric acid, cysteins,
thioglycerol, thioglycolic acid, thiosorbitol, butylated
hydroxanisol, butylated hydroxyltoluene, and propyl gallate, and
mixtures thereof); agents to maintain isotonicity (e.g., sodium
chloride, sugars, polyols (sugar alcohols), boric acid, sodium
tartrate, propylene glycol, and mixtures thereof); one or more
sugars (e.g., trehalose, maltose, sucrose, lactose, mannose,
dextrose, fructose, etc.) or sugar alcohol (e.g., sorbitol,
maltitol, lactitol, mannitol, glycerol, etc.); alcohol (e.g.,
ethanol, t-butanol, etc.); and preservatives (alcohols, benzoic
acid, salicylic acid, phenol and its derivatives (e.g., cresol,
p-cresol, m-cresol and o-cresol), cetrimide, BHA (butylated
hydroxytoluene), BHA (butylated hydroxyanisole); and mixtures
thereof).
[0184] pH adjusting agents useful in the practice of the present
invention include, but are not limited to hydrochloric acid, sodium
hydroxide, citric acid, phthalic acid, acetic acid, ascorbic acid,
phosphate, glutamate, sodium or potassium succinate, tartrate,
histidine, sodium or potassium phosphate, Tris (tris
(hydroxymethyl)aminomethane), diethanolamine, sulfuric acid, and
phosphoric acid. Buffers comprising both acids and bases/salts can
also be used. Preferred pH adjusting agents comprise sulfuric acid
and phosphoric acid.
[0185] In a preferred embodiment of the present invention, the
liposomes can be rehydrated using buffered aqueous solutions (e.g.,
phosphate buffer saline (PBS)), 0.9% Saline, 5% Dextrose, 10%
Sucrose, or water for injection (WFI) as the rehydration medium).
In some embodiments, the pH of the aqueous phase of the liposomal
compositions is adjusted, for example, to approximately human
physiological pH (i.e., between about pH 6.5 and about pH 7.5).
Excipients typically present in the aqueous phase include, but are
not limited to, buffer systems, agents to maintain isotonicity,
sugars, sugar alcohols, and/or preservatives.
[0186] Exemplary embodiments of liposomal compositions of peptide
epoxyketones include, but are not limited to, the following:
carfilzomib-EPC; carfilzomib-EPC-cholesterol; carfilzomib-DSPC;
carfilzomib-DSPC-cholesterol; carfilzomib-DPPC;
carfilzomib-DPPC-cholesterol; carfilzomib-sphingomyelin;
carfilzomib-sphingomyelin-cholesterol.
[0187] Further examples comprise PEGylated liposomal compositions
of peptide epoxyketones that include, but are not limited to, the
following: carfilzomib-EPC-mPEG2000DSPE;
carfilzomib-EPC-mPEG2000DSPE-cholesterol;
carfilzomib-DSPC-mPEG2000DSPE;
carfilzomib-DSPC-mPEG2000DSPE-cholesterol;
carfilzomib-sphingomyelin-mPEG2000DSPE; and
carfilzomib-sphingomyelin-mPEG2000DSPE-cholesterol.
[0188] Preferred embodiments of liposomal compositions of peptide
epoxyketones include, but are not limited to, the following:
carfilzomib-EPC-mPEG2000DSPE-cholesterol;
carfilzomib-sphingomyelin-mPEG2000DSPE-cholesterol; and
carfilzomib-HSPC-mPEG2000DSPE-cholesterol.
[0189] Examples of embodiments of liposomal compositions of the
present invention are set forth in Examples 1, 7, 10, and 11.
Further, examples of ranges of weight percents and ratios for
drug:total lipid are presented in Table I; and examples of ranges
of total lipid weight percents and total lipid weight ratios are
presented in Table II. Accordingly, additional examples of
preferred embodiments of liposomal compositions of the present
invention include selecting a drug:total lipid combination from
Table I and combining it with a total lipid combination from Table
II (see, e.g., Example 1, Table 3).
TABLE-US-00001 TABLE I Examples of Drug:Total Lipid Combinations
Drug Weight Percent Range Drug:Total Lipid Total Lipid Weight
Percent Range Weight Ratio Range between about 0.5 wt. % and about
50 wt. % about 0.005:0.995 to peptide epoxyketone compound about
0.5:0.5 between about 99.5 wt. % and about 50 wt. % total lipid
between about 0.5 wt. % and about 35 wt. % about 0.005:0.995 to
peptide epoxyketone compound about 0.35:0.65 between about 99.5 wt.
% and about 65 wt. % total lipid between about 1 wt. % and about 30
wt. % about 0.01:0.99 to peptide epoxyketone compound about
0.30:0.70 between about 99 wt. % and about 70 wt. % total lipid
between about 1 wt. % and about 25 wt. % about 0.01:0.99 to peptide
epoxyketone compound about 0.25:0.75 between about 99 wt. % and
about 75 wt. % total lipid
TABLE-US-00002 TABLE II Examples of Total Lipid Combinations Total
Lipid Weight Ratio Range (Phospholipid:Hydrophilic
Polymer-derivatized Lipid:{Cholesterol or Lipid Weight Percent
Range Cholesterol Derivative}) Phospholipid 100 wt. % 1:0:0
Phospholipid & between about 90 wt. % phospholipid about
0.9:0:0.1 {Cholesterol or & about 50 wt. % phospholipid to
about 0.5:0:0.5 Cholesterol between about 10 wt. % cholesterol or
Derivative} derivative and about 50 wt. % cholesterol or derivative
Phospholipid & between about 90 wt. % phospholipid about
0.9:0.1:0 Hydrophilic & about 75 wt. % phospholipid to about
0.75:0.25:0 Polymer- between about 10 wt. % hydrophilic derivatized
Lipid polymer-derivatized lipid & about 25 wt. % hydrophilic
polymer-derivatized lipid Phospholipid, between about 83.3 wt. %
about 0.833:0.0833:0.0833 Hydrophilic phospholipid & about 57
wt. % to about 0.57:0.14:0.29 Polymer- phospholipid derivatized
between about 8.33 wt. % hydrophilic Lipid, &
polymer-derivatized lipid & about 14 wt. % {Cholesterol or
hydrophilic polymer-derivatized Cholesterol lipid Derivative}
between about 8.33 wt. % cholesterol or derivative & about 29
wt. % cholesterol or derivative
4.0.0 PREPARING LIPOSOMAL COMPOSITIONS
[0190] Liposomes can be prepared by a variety of techniques (e.g.,
Szoka, F., Jr., et al., "Comparative Properties and Methods of
Preparation of Lipid Vesicles (Liposomes)," Annual Review of
Biophysics and Bioengineering, June 1980, 9:467-508; U.S. Pat. No.
4,235,871) including reverse phase evaporation methods. The reverse
phase evaporation vesicles initially have typical average sizes
between about 2-4 microns.
[0191] In some embodiments, liposomes are formed by simple
lipid-film hydration techniques (see, e.g., Examples 1 and 2). In
this procedure, a mixture of liposome-forming lipids of the type
described herein and peptide expoxyketone compounds are dissolved
in a suitable organic solvent and evaporated in a vessel to form a
thin film, which is then covered by an aqueous medium. The lipid
film hydrates to form vesicles typically with sizes between about
0.1 to 10 microns.
[0192] Other embodiments of the present invention include, a method
of passively encapsulating a hydrophobic, water-insoluble, peptide
expoxyketone compound into the internal aqueous core of the
liposome. Such encapsulation in the aqueous core can be facilitated
using one or more solubilizing agent. Solubilizing agents increase
the solubility of a peptide expoxyketone compound in an aqueous
solution. Solubilizing agents include, for example, compounds to
facilitate solubilization (e.g., cyclodextrin), pH adjusting
agents, cosolvents, and combinations thereof. Advantages of
encapsulating peptide expoxyketone compounds in the interior
aqueous core of liposomes include greater protection from chemical
and biological degradation, slower diffusion, and extended drug
release profiles. Further, as described below in the Experimental
Section, the liposomal compositions comprising peptide epoxyketone
compounds of the present invention enhance the therapeutic window
of peptide epoxyketone compounds by: improving in vivo half-life
relative to non-liposomal compositions comprising peptide
epoxyketone compounds; providing desirable pharmacodynamic
profiles; and providing anti-tumor activity in a human tumor
xenograft model greater than or equal to non-liposomal compositions
comprising peptide epoxyketone compounds. Further, the liposomal
compositions of the present invention demonstrated improved
tolerability of liposomal compositions comprising peptide
epoxyketone compounds relative to non-liposomal compositions
comprising peptide epoxyketone compounds.
[0193] Cyclodextrins are an example of compounds to facilitate
solubilization of peptide expoxyketone compounds in aqueous
solution. Cyclodextrins can be charged or neutral, native
(cyclodextrins .alpha., .beta., .gamma., .delta., .epsilon.),
branched or polymerized. In certain aspects, cyclodextrins can be
chemically modified, for example, by substitution of one or more
hydroxypropyls by groups such as alkyls, aryls, arylalkyls,
glycosidics, or by etherification, esterification with alcohols or
aliphatic acids. From these groups, particular preference is given
to those from hydroxypropyl, methyl, and sulfobutylether groups
(see, e.g., Stella V. J., et al., Toxicol. Pathol. 36(1):30-42
(2008)). In certain aspects, cyclodextrins comprise six, seven, or
eight glucopyranose units.
[0194] Cyclodextrins include .alpha.-cyclodextrin,
.beta.-cyclodextrin, and .gamma.-cylcodextrin. Suitable
.alpha.-cyclodextrins include but are not limited to
hydroxypropyl-.alpha.-cyclodextrin and
hydroxyethyl-.alpha.-cyclodextrin. Suitable .beta.-cyclodextrins
include but are not limited to hydroxypropyl-.beta.-cyclodextrin
(e.g., 2-hydroxypropyl cyclodextrin),
carboxymethyl-.beta.-cyclodextrin,
dihydroxypropyl-.beta.-cyclodextrin,
hydroxyethyl-.beta.-cyclodextrin,
2,6-di-O-methyl-.beta.-cyclodextrin, methyl-.beta.-cyclodextrin,
randomly methylated cylcodextrin, and sulfated-.beta.-cyclodextrin.
Suitable .gamma.-cyclodextrins include hydroxypropyl
.gamma.-cyclodextrin, dihydroxypropyl-.gamma.-cyclodextrin,
hydroxyethyl .gamma.-cyclodextrin, and
sulfated-.gamma.-cyclodextrin.
[0195] Preferred cyclodextrins for use in the practice of the
present invention include .beta.-cyclodextrins (such as sulfobutyl
ether-.beta.-cyclodextrins (abbreviated as SBE-.beta.-CD or
SBE-B-CD; e.g., CAPTISOL.RTM. (Ligand Pharmaceuticals, Inc., La
Jolla, Calif.), see also U.S. Pat. Nos. 4,535,152; 4,727,064;
5,134,127; 5,173,481); or hydroxypropyl-betacyclodextrin (HP-13-CD;
Janssen, Titusville N.J.; see also Gould S, et al., Food Chem.
Toxicol. 43(10):1451-9 (2005)); see also U.S. Pat. Nos. 4,920,214;
5,385,891; 5,718,905; and 6,046,177).
[0196] Peptide expoxyketone compounds are often hydrophobic and
have low solubility in water. Peptide expoxyketone compounds have
increased aqueous solubility in acidic solutions. Accordingly,
lowering the pH of the aqueous solution in which a peptide
expoxyketone compound is being dissolved can enhance aqueous
solubilization. For example, the pH of the aqueous solution can be
lowered using a pH adjusting agent to a pH of between about pH 0.5
and about pH 3, preferably to a pH of between about pH 0.5 and
about pH 2 using an acid, for example, hydrochloric acid. Examples
of pH adjusting agents are listed above. Preferred pH adjusting
agents for solubilization of peptide epoxyketone compounds include,
but are not limited to, hydrochloric acid, citric acid,
methanesulfonic acid, sulfuric acid, tartaric acid, acetic acid,
phosphoric acid, and/or maleic acid. A preferred pH for
solubilization is typically between about pH 1 and about pH 2.
[0197] Further, solubility of peptide expoxyketone compounds in
aqueous solutions can be increased by the use of cosolvent
solubilization. Examples of cosolvents as solubilizing agents
include, but are not limited to, dimethylsulfoxide,
methylpyrrolidone, dimethylimidazolidinone, tetrahydrofuran,
N,N-dimethylacetamide, propylene glycol, benzyl alcohol,
polyethylene glycol, ethanol, methanol, isopropyl alcohol,
dimethylformamide, and combinations thereof. Preferred cosolvents
include dimethylsulfoxide, methylpyrrolidone, propylene glycol,
polyethylene glycol, ethanol, methanol, isopropyl alcohol,
dimethylformamide, and combinations thereof.
[0198] As noted above, solubility of peptide expoxyketone compounds
in aqueous solutions can be increased by use of solubilizing
agents, including, but not limited to, compounds, pH adjusting
agents, cosolvents, and combinations thereof.
[0199] Metals and metal ions can also be used to facilitate loading
of drug into liposomes (see, e.g., WO/2003/028697 and U.S. Pat.
Nos. 5,466,467; 5,663,387; and 5,837,282). Such metals and metal
ions include, but are not limited to, divalent metal cations and
transition metals (e.g., Mn, Ca, Fe, Co, Ni, Cu, Zn, V, Ti, Cr, Rh,
Ru, Mo, and Pd). Drug can be stably entrapped within transition
metal-containing liposomes, typically as a result of metal/drug
complexation (see, e.g., Ramsay E., et al., Pharm Res.
23(12):2799-808 (2006)).
[0200] In some embodiments, liposomes are formed by a thin film
hydration method followed by rehydration using an aqueous solution
comprising a peptide expoxyketone compound and solubilizing agent.
In such a method, a lipid film is formed wherein the lipid film
comprises, for example, any one or combination of lipids, including
but not limited to the following: L-.alpha.-phosphatidylcholine
(e.g., egg phosphatidylcholine (EPC), or hydrogenated soy
phosphatidylcholine (HSPC));
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC);
1,2-Distearoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DSPG);
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC);
sphingomyelin (SPH);
1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE);
phospholipids conjugated to monomethoxy polyethylene glycol (mPEG);
and cholesterol. The lipids are typically dissolved in an organic
solvent (e.g., Methanol:Chloroform) followed by solvent removal to
form a lipid film.
[0201] The peptide expoxyketone compound is solubilized in an
aqueous solution comprising, for example, about 1% to about 60%
(w/w), preferably about 5% to about 40% of a solubilizing agent
(e.g., sulfobutylether-betacyclodextrin or
hydroxypropyl-betacyclodextrin). The aqueous solution can also
include, for example, a pH adjusting agent (e.g., citrate buffer,
.about.pH 3, or Glycine-HCl; .about.pH 2) and/or a cosolvent for
solubilization of the peptide expoxyketone compound. The aqueous
drug solution is used to rehydrate the lipid film. Upon
rehydration, self-assembling vesicles form concentric lipid
bilayers encapsulating an internal aqueous volume (i.e., aqueous
core) of the aqueous solution comprising the peptide expoxyketone
compound. The unencapsulated free drug can be removed, for example,
by centrifugation and the liposomal composition washed, for
example, with phosphate buffer saline. Example 7 describes making
liposomal compositions following this method.
[0202] In other embodiments, liposomes are formed by a lipid
solution injection method wherein a lipid solution is injected into
an aqueous solution comprising a peptide expoxyketone compound.
This method typically comprises solubilizing a peptide expoxyketone
compound (e.g., in different solid states, such as crystalline or
amorphous), using a solubilizing agent (e.g., pH control, with or
without, cosolvent solubilization) in an aqueous solution. The
lipids are dissolved in a solvent, for example, an organic solvent
(such as an alcohol (e.g., ethanol) or an ether), followed by
injection into the aqueous solution comprising the peptide
expoxyketone compound while stirring. Liposome vesicles are formed
upon injection into the aqueous solution trapping small amounts of
aqueous solution in the internal aqueous compartment(s) of the
vesicles. Example 10 describes making liposomal compositions
following this method. One advantage of this method is that it is
scalable.
[0203] In some embodiments, for example for pH adjustment and/or
removal of solvent and/or a cosolvent, the methods of the invention
further comprise processing the liposomal composition using
dialysis, desalting, buffer exchange, and/or gel filtration.
[0204] A liposomal composition of the present invention generally
contains a non-homogenous mixture of lipids, peptide epoxyketone
compound, and aqueous solution, wherein the liposomes are of
substantially homogenous size, with an average size of less than
about 1 micron, preferably between about 0.01 microns to about 1.0
micron, preferably between about 0.05 microns and about 0.5
microns, between about 0.05 microns and about 0.20 microns, between
about 0.05 microns and about 0.15 microns, and preferably between
about 0.05 microns and about 0.10 microns. In some embodiments,
liposomes of the liposomal compositions of the present invention
have average diameters of less than about 0.2 microns. Sizing
serves to eliminate larger liposomes and to produce a defined size
range having optimal pharmacokinetic properties.
[0205] One effective sizing method for vesicles involves extruding
an aqueous suspension of the liposomes through a series of
polycarbonate membranes having a selected uniform pore size in the
range of 0.03 to 0.2 micron, typically 0.05, 0.08, 0.1, or 0.2
microns. The pore size of the membrane corresponds roughly to the
largest sizes of liposomes produced by extrusion through that
membrane, particularly where the preparation is extruded two or
more times through the same membrane. The liposomes can be extruded
through successively smaller-pore membranes, to achieve a gradual
reduction in liposome size. This method of liposome sizing is used
in preparing homogeneous-size vesicle compositions. A more recent
method involves extrusion through an asymmetric ceramic filter
(see, e.g., U.S. Pat. No. 4,737,323). Homogenization methods are
also useful for down-sizing liposomes to sizes of 0.1 micron or
less.
[0206] Sonicating a liposome suspension either by bath or probe
sonication can be used to produce progressive size reduction down
to small unilamellar vesicles (SUVs) less than about 0.05 microns
in size. Homogenization is another method that relies on shearing
energy to fragment large liposomes into smaller ones. In a typical
homogenization procedure, vesicles are recirculated through a
standard emulsion homogenizer until selected liposome sizes,
typically between about 0.1 and 0.5 microns, are observed. In both
methods, the particle size distribution can be monitored by
conventional laser-beam particle size discrimination. A further
sizing method includes use of a microfluidizer.
[0207] Centrifugation and molecular sieve chromatography are other
methods available for producing a liposome suspension with particle
sizes below a selected threshold less than 1 micron. These two
methods both involve preferential removal of larger liposomes,
rather than conversion of large particles to smaller ones.
[0208] Examples of preparation, rehydration, and characterization
of liposomal compositions of the present invention are presented in
Example 1, Example 2, Example 3, Example 7, Example 10, and Example
11 herein.
[0209] In one aspect, the present invention includes methods for
the preparation of the liposomal compositions described herein. In
one embodiment, a method of making a liposomal composition
comprises mixing (typically dissolving) lipid and peptide
epoxyketone compound in a suitable solvent, evaporating the solvent
to produce a dried film, rehydrating the dried film (which in this
embodiment comprises lipid and peptide epoxyketone compound) to
form liposomes, and sizing the liposomes. In another embodiment, a
method of making a liposomal composition comprises a thin film
hydration method which produces a dried film comprising liposomal
components followed by rehydration using an aqueous solution
comprising a peptide expoxyketone compound as well as a
solubilizing agent (e.g., a pH adjusting agent, and/or a
cosolvent). In yet another embodiment, a method of making a
liposomal composition comprises dissolving lipid(s) in solvent(s)
and injecting the resulting lipid solution into an aqueous solution
comprising a peptide expoxyketone compound as well as a
solubilizing agent (e.g., a pH adjusting agent, and/or a
cosolvent). In yet another embodiment, a remote loading method
(using, e.g., pH-gradient loading; see, e.g., Avnir, Y., et al.,
Arthritis & Rheumatism, 58(1):119-129 (2008); {hacek over
(C)}eh, B., et al., Journal of Colloid and Interface Science,
185(1): 9-18 (1997); Vemuri S, et al., J. Pharm. Pharmacol.,
46(10):778-83 (1994); Dos Santos, N., et al., Biochimica et
Biophysica Acta, 1661:47-60 (2004)) for loading drug into liposomes
is used to prepare the liposomal compositions described herein.
Remote loading methods typically produce higher drug loading into
liposomes compared to thin-film rehydration methods.
[0210] The present invention also includes liposomal compositions
comprising peptide expoxyketone compounds made by the methods
described herein.
[0211] Dry pharmaceutical compositions comprising one or more
lipids and a peptide epoxyketone compound can be formed by drying
the liposomal compositions described herein, for example, by
lyophilization, desiccation, freeze-drying, spray-drying, or
similar method. In some embodiments, dry pharmaceutical
compositions further comprise additional excipients, for example
cryoprotectant agents (e.g., glycerol, dimethylamine,
dimethylsulfoxide), glass transition modifying agents (e.g. sugars,
polyols, polymers, amino acids), and/or other stabilizing
excipients. Such dry pharmaceutical compositions can be rehydrated
for use in the methods of the present invention. The rehydration
media used for reconstitution of such dry pharmaceutical
compositions can include excipients including, but not limited to,
a pH adjusting agent, an antioxidant, an agent to maintain
isotonicity, a sugar, a sugar alcohol, an alcohol, and/or a
preservative.
5.0.0 USES OF THE LIPOSOMAL COMPOSITIONS OF THE PRESENT
INVENTION
[0212] The biological consequences of proteasome inhibition are
numerous. Proteasome inhibition has been suggested as a prevention
and/or treatment of a multitude of diseases including, but not
limited to, proliferative diseases, neurotoxic/degenerative
diseases, Alzheimer's, ischemic conditions, inflammation,
auto-immune diseases, HIV, cancers, organ graft rejection, septic
shock, inhibition of antigen presentation, decreasing viral gene
expression, parasitic infections, conditions associated with
acidosis, macular degeneration, pulmonary conditions, muscle
wasting diseases, fibrotic diseases, bone and hair growth diseases.
Therefore, pharmaceutical formulations for very potent,
proteasome-specific compounds, such as the epoxy ketone class of
molecules, provide a means of administering a drug to a subject and
treating these conditions.
[0213] At the cellular level, the accumulation of polyubiquitinated
proteins, cell morphological changes, and apoptosis have been
reported upon treatment of cells with various proteasome
inhibitors. Proteasome inhibition has also been suggested as a
possible antitumor therapeutic strategy. The fact that epoxomicin
was initially identified in a screen for antitumor compounds
validates the proteasome as an antitumor chemotherapeutic target.
Accordingly, these liposomal compositions are useful for treating
cancer.
[0214] Both in vitro and in vivo models have shown that malignant
cells, in general, are susceptible to proteasome inhibition. In
fact, proteasome inhibition has already been validated as a
therapeutic strategy for the treatment of multiple myeloma. This
could be due, in part, to the highly proliferative malignant cell's
dependency on the proteasome system to rapidly remove proteins
(Rolfe, et al., J. Mol. Med. 75:5-17 (1997); Adams, Nature 4:
349-360 (2004)). Therefore, provided herein is a method of treating
cancers comprising administering to a subject in need of such
treatment a therapeutically effective amount of a liposomal
composition of a peptide expoxyketone compound as provided
herein.
[0215] Cancer refers to diseases of blood, bone, organs, skin
tissue and the vascular system, including, but not limited to,
cancers of the bladder, blood, bone, brain, breast, cervix, chest,
colon, endrometrium, esophagus, eye, head, kidney, liver, lung,
lymph nodes, mouth, neck, ovaries, pancreas, prostate, rectum,
renal, skin, stomach, testis, throat, and uterus. Specific cancers
include, but are not limited to, leukemia (acute lymphocytic
leukemia (ALL), acute myelogenous leukemia (AML), chronic
lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML),
hairy cell leukemia, mature B cell neoplasms (small lymphocytic
lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic
lymphoma (such as Waldenstrom's macroglobulinemia), splenic
marginal zone lymphoma, plasma cell myeloma, plasmacytoma,
monoclonal immunoglobulin deposition diseases, heavy chain
diseases, extranodal marginal zone B cell lymphoma (MALT lymphoma),
nodal marginal zone B cell lymphoma (NMZL), follicular lymphoma,
mantle cell lymphoma, diffuse B cell lymphoma, mediastinal (thymic)
large B cell lymphoma, intravascular large B cell lymphoma, primary
effusion lymphoma and Burkitt lymphoma/leukemia), mature T cell and
natural killer (NK) cell neoplasms (T cell prolymphocytic leukemia,
T cell large granular lymphocytic leukemia, aggressive NK cell
leukemia, adult T cell leukemia/lymphoma, extranodal NK/T cell
lymphoma, enteropathy-type T cell lymphoma, hepatosplenic T cell
lymphoma, blastic NK cell lymphoma, mycosis fungoides (Sezary
syndrome), primary cutaneous anaplastic large cell lymphoma,
lymphomatoid papulosis, angioimmunoblastic T cell lymphoma,
unspecified peripheral T cell lymphoma and anaplastic large cell
lymphoma), Hodgkin lymphoma (nodular sclerosis, mixed celluarity,
lymphocyte-rich, lymphocyte depleted or not depleted, nodular
lymphocyte-predominant), myeloma (multiple myeloma, indolent
myeloma, smoldering myeloma), chronic myeloproliferative disease,
myelodysplastic/myeloproliferative disease, myelodysplastic
syndromes, immunodeficiency-associated lymphoproliferative
disorders, histiocytic and dendritic cell neoplasms, mastocytosis,
chondrosarcoma, Ewing sarcoma, fibrosarcoma, malignant giant cell
tumor, myeloma bone disease, osteosarcoma, breast cancer (hormone
dependent, hormone independent), gynecological cancers (cervical,
endometrial, fallopian tube, gestational trophoblastic disease,
ovarian, peritoneal, uterine, vaginal and vulvar), basal cell
carcinoma (BCC), squamous cell carcinoma (SCC), malignant melanoma,
dermatofibrosarcoma protuberans, Merkel cell carcinoma, Kaposi's
sarcoma, astrocytoma, pilocytic astrocytoma, dysembryoplastic
neuroepithelial tumor, oligodendrogliomas, ependymoma, glioblastoma
multiforme, mixed gliomas, oligoastrocytomas, medulloblastoma,
retinoblastoma, neuroblastoma, germinoma, teratoma, malignant
mesothelioma (peritoneal mesothelioma, pericardial mesothelioma,
pleural mesothelioma), gastro-entero-pancreatic or
gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid,
pancreatic endocrine tumor (PET), colorectal adenocarcinoma,
colorectal carcinoma, aggressive neuroendocrine tumor,
leiomyosarcomamucinous adenocarcinoma, Signet Ring cell
adenocarcinoma, hepatocellular carcinoma, cholangiocarcinoma,
hepatoblastoma, hemangioma, hepatic adenoma, focal nodular
hyperplasia (nodular regenerative hyperplasia, hamartoma),
non-small cell lung carcinoma (NSCLC) (squamous cell lung
carcinoma, adenocarcinoma, large cell lung carcinoma), small cell
lung carcinoma, thyroid carcinoma, prostate cancer (hormone
refractory, androgen independent, androgen dependent,
hormone-insensitive), and soft tissue sarcomas (fibrosarcoma,
malignant fibrous hystiocytoma, dermatofibrosarcoma, liposarcoma,
rhabdomyosarcoma leiomyosarcoma, hemangiosarcoma, synovial sarcoma,
malignant peripheral nerve sheath tumor/neurofibrosarcoma,
extraskeletal osteosarcoma).
[0216] In some embodiments, a liposomal composition comprising a
peptide expoxyketone compound as provided herein, or a
pharmaceutical composition comprising the same, can be administered
to treat multiple myeloma in a subject. For example, multiple
myeloma can include refractory and/or refractory multiple
myeloma.
[0217] Many tumors of the haematopoietic and lymphoid tissues are
characterized by an increase in cell proliferation, or a particular
type of cell. The chronic myeloproliferative diseases (CMPDs) are
clonal haematopoietic stem cell disorders characterized by
proliferation in the bone marrow of one or more of the myeloid
lineages, resulting in increased numbers of granulocytes, red blood
cells and/or platelets in the peripheral blood. As such, use of a
proteasome inhibitor for the treatment of such diseases is
attractive and being examined (Cilloni, et al., Haematologica 92:
1124-1229 (2007)). CMPD can include chronic myelogenous leukemia,
chronic neutrophilic leukemia, chronic eosinophilic leukemia,
polycythaemia vera, chronic idiopathic myelofibrosis, essential
thrombocythaemia and unclassifiable chronic myeloproliferative
disease. Provided herein is a method of treating CMPD comprising
administering to a subject in need of such treatment a
therapeutically effective amount of the liposomal compositions
comprising peptide epoxyketone compounds disclosed herein.
[0218] Myelodisplastic/myeloproliferative diseases, such as chronic
myelomonocytic leukemia, atypical chronic myeloid leukemia,
juvenile myelomonocytic leukemia and unclassifiable
myelodysplastic/myeloproliferative disease, are characterized by
hypercellularity of the bone marrow due to proliferation in one or
more of the myeloid lineages Inhibiting the proteasome with a
liposomal composition comprising a peptide epoxyketone compound
described herein, can serve to treat these
myelodisplatic/myeloproliferative diseases by providing a subject
in need of such treatment a therapeutically effective amount of the
liposomal composition.
[0219] Myelodysplastic syndromes (MDS) refer to a group of
hematopoietic stem cell disorders characterized by dysplasia and
ineffective haematopoiesis in one or more of the major myeloid cell
lines. Targeting NF-kB with a proteasome inhibitor in these
hematologic malignancies induces apoptosis, thereby killing the
malignant cell (Braun, et al., Cell Death and Differentiation
13:748-758 (2006)). Further provided herein is a method to treat
MDS comprising administering to a subject in need of such treatment
a therapeutically effective amount of a liposomal composition
comprising a peptide epoxyketone compound provided herein. MDS
includes refractory anemia, refractory anemia with ringed
sideroblasts, refractory cytopenia with multilineage dysplasia,
refractory anemia with excess blasts, unclassifiable
myelodysplastic syndrome, and myelodysplastic syndrome associated
with isolated del (5q) chromosome abnormality.
[0220] Mastocytosis is a proliferation of mast cells and their
subsequent accumulation in one or more organ systems. Mastocytosis
includes, but is not limited to, cutaneous mastocytosis, indolent
systemic mastocytosis (ISM), systemic mastocytosis with associated
clonal haematological non-mast-cell-lineage disease (SM-AHNMD),
aggressive systemic mastocytosis (ASM), mast cell leukemia (MCL),
mast cell sarcoma (MCS) and extracutaneous mastocytoma. Further
provided herein is a method to treat mastocytosis comprising
administering a therapeutically effective amount of the compound
disclosed herein to a subject diagnosed with mastocytosis.
[0221] The proteasome regulates NF-.kappa.B, which in turn
regulates genes involved in the immune and inflammatory response.
For example, NF-.kappa.B is required for the expression of the
immunoglobulin light chain .kappa. gene, the IL-2 receptor
.alpha.-chain gene, the class I major histocompatibility complex
gene, and a number of cytokine genes encoding, for example, IL-2,
IL-6, granulocyte colony-stimulating factor, and IFN-.beta.
(Palombella, et al., Cell 78:773-785 (1994)). Thus, provided herein
are methods of affecting the level of expression of IL-2, MHC-I,
IL-6, TNF.alpha., IFN-.beta. or any of the other
previously-mentioned proteins, each method comprising administering
to a subject a therapeutically effective amount of a liposomal
composition comprising a peptide expoxyketone compound as disclosed
herein.
[0222] Also provided herein is a method of treating an autoimmune
disease in a subject comprising administering a therapeutically
effective amount of a liposomal composition of a peptide
expoxyketone compound described herein. Examples of autoimmune
diseases or disorders include, but are not limited to, inflammatory
responses such as inflammatory skin diseases including psoriasis
and dermatitis (e.g. atopic dermatitis); systemic scleroderma and
sclerosis; responses associated with inflammatory bowel disease
(such as Crohn's disease and ulcerative colitis); respiratory
distress syndrome (including adult respiratory distress syndrome
(ARDS)); dermatitis; meningitis; encephalitis; uveitis; colitis;
glomerulonephritis; allergic conditions such as eczema and asthma
and other conditions involving infiltration of T cells and chronic
inflammatory responses; atherosclerosis; leukocyte adhesion
deficiency; rheumatoid arthritis; systemic lupus erythematosus
(SLE); diabetes mellitus (e.g. Type I diabetes mellitus or insulin
dependent diabetes mellitus); multiple sclerosis; Reynaud's
syndrome; autoimmune thyroiditis; allergic encephalomyelitis;
Sjorgen's syndrome; juvenile onset diabetes; and immune responses
associated with acute and delayed hypersensitivity mediated by
cytokines and T-lymphocytes typically found in tuberculosis,
sarcoidosis, polymyositis, granulomatosis and vasculitis;
pernicious anemia (Addison's disease); diseases involving leukocyte
diapedesis; central nervous system (CNS) inflammatory disorder;
multiple organ injury syndrome; hemolytic anemia (including, but
not limited to cryoglobinemia or Coombs positive anemia);
myasthenia gravis; antigen-antibody complex mediated diseases;
anti-glomerular basement membrane disease; antiphospholipid
syndrome; allergic neuritis; Graves' disease; Lambert-Eaton
myasthenic syndrome; pemphigoid bullous; pemphigus; autoimmune
polyendocrinopathies; Reiter's disease; stiff-man syndrome; Beheet
disease; giant cell arteritis; immune complex nephritis; IgA
nephropathy; IgM polyneuropathies; and immune thrombocytopenic
purpura (ITP) or autoimmune thrombocytopenia.
[0223] The immune system screens for autologous cells that are
virally infected, have undergone oncogenic transformation, or
present unfamiliar peptides on their surface. Intracellular
proteolysis generates small peptides for presentation to
T-lymphocytes to induce MHC class I-mediated immune responses.
Thus, provided herein is a method of using a liposomal composition
comprising a peptide epoxyketone compound provided herein as an
immunomodulatory agent for inhibiting or altering antigen
presentation in a cell, comprising exposing the cell (or
administering to a subject) to the compound described herein.
Specific embodiments include a method of treating graft or
transplant-related diseases, such as graft-versus-host disease or
host versus-graft disease in a subject, comprising administering a
therapeutically effective amount of the compound described herein.
Grafts include such diverse material as, for example, isolated
cells such as islet cells; tissue such as the amniotic membrane of
a newborn, bone marrow, hematopoietic precursor cells, and ocular
tissue, such as corneal tissue; and organs such as skin, heart,
liver, spleen, pancreas, thyroid lobe, lung, kidney, tubular organs
(e.g., intestine, blood vessels, or esophagus). The tubular organs
can be used to replace damaged portions of esophagus, blood
vessels, or bile duct. The skin grafts can be used not only for
burns, but also as a dressing to damaged intestine or to close
certain defects such as diaphragmatic hernia. The graft is derived
from any mammalian source, including human, whether from cadavers
or living donors. In some cases, the donor and recipient is the
same subject. In some embodiments, the graft is bone marrow or an
organ such as heart and the donor of the graft and the host are
matched for HLA class II antigens.
[0224] Histiocytic and dendritic cell neoplasms are derived from
phagocytes and accessory cells, which have major roles in the
processing and presentation of antigens to lymphocytes. Depleting
the proteasome content in dendritic cells has been shown to alter
their antigen-induced responses (Chapatte, et al., Cancer Res.
(2006) 66:5461-5468). In some embodiments, a liposomal composition
comprising a peptide expoxyketone compound provided herein can be
administered to a subject with histiocytic or dendritic cell
neoplasm. Histiocytic and dendritic cell neoplasms include
histiocytic sarcoma, Langerhans cell histiocytosis, Langerhans cell
sarcoma, interdigitating dendritic cell sarcoma/tumor, follicular
dendritic cell sarcoma/tumor and non-specified dendritic cell
sarcoma.
[0225] Inhibition of the proteasome has been shown to be beneficial
to treat diseases whereby a cell type is proliferating and immune
disorders; thus, in some embodiments, the treatment of
lymphoproliferative diseases (LPD) associated with primary immune
disorders (PID) is provided comprising administering a
therapeutically effective amount of a liposomal composition
comprising a peptide epoxyketone compound to a subject in need
thereof. The most common clinical settings of immunodeficiency
associated with an increased incidence of lymphoproliferative
disorders, including B-cell and T-cell neoplasms and lymphomas, are
primary immunodeficiency syndromes and other primary immune
disorders, infection with the human immunodeficiency virus (HIV),
iatrogenic immunosuppression in subjects who have received solid
organ or bone marrow allografts, and iatrogenis immunosuppression
associated with methotrexate treatment. Other PIDs commonly
associated with LPDs, but not limited to, are ataxia telangiectasia
(AT), Wiskott-Aldrich syndrome (WAS), common variable
immunodeficiency (CVID), severe combined immunodeficiency (SCID),
X-linked lymphoproliferative disorder (XLP), Nijmegen breakage
syndrome (NBS), hyper-IgM syndrome, and autoimmune
lymphoproliferative syndrome (ALPS).
[0226] Proteasome inhibition has also been associated with
inhibition of NF-.kappa.B activation and stabilization of p53
levels. Thus, compositions provided herein may also be used to
inhibit NF-.kappa.B activation, and stabilize p53 levels in cell
culture. Since NF-.kappa.B is a key regulator of inflammation, it
is an attractive target for anti-inflammatory therapeutic
intervention. Thus, liposomal compositions comprising peptide
epoxyketone compounds provided herein may be useful for the
treatment of conditions associated with inflammation, including,
but not limited to COPD, psoriasis, asthma, bronchitis, emphysema,
and cystic fibrosis.
[0227] The disclosed liposomal compositions comprising peptide
epoxyketone compounds can be used to treat conditions mediated
directly by the proteolytic function of the proteasome such as
muscle wasting, or mediated indirectly via proteins that are
processed by the proteasome such as NF-.kappa.B. The proteasome
participates in the rapid elimination and post-translational
processing of proteins (e.g., enzymes) involved in cellular
regulation (e.g., cell cycle, gene transcription, and metabolic
pathways), intercellular communication, and the immune response
(e.g., antigen presentation). Specific examples discussed below
include .beta.-amyloid protein and regulatory proteins such as
cyclins and transcription factor NF-.kappa.B.
[0228] In one embodiment of the present invention, the liposomal
compositions comprising peptide epoxyketone compounds are useful
for the treatment of cancer. Compounds of the invention also can be
used to inhibit NF-.kappa.B activation, and stabilize p53 levels in
cell culture.
[0229] In one embodiment of the present invention, the liposomal
compositions can be used for anti-inflammatory therapeutic
intervention in treating conditions associated with chronic
inflammation, including, but not limited to COPD, psoriasis,
bronchitis, emphysema, and cystic fibrosis.
[0230] In another embodiment of the present invention, the
liposomal compositions can be used to treat neurodegenerative
diseases and conditions, including, but not limited to: stroke;
ischemic damage to the nervous system; neural trauma (e.g.,
percussive brain damage, spinal cord injury, and traumatic damage
to the nervous system); multiple sclerosis and other
immune-mediated neuropathies (e.g., Guillain-Barre syndrome and its
variants, acute motor axonal neuropathy, acute inflammatory
demyelinating polyneuropathy, and Fisher Syndrome); HIV/AIDS
dementia complex; axonomy; diabetic neuropathy; Parkinson's
disease; Huntington's disease; multiple sclerosis; bacterial,
parasitic, fungal, and viral meningitis; encephalitis; vascular
dementia; multi-infarct dementia; Lewy body dementia; frontal lobe
dementia such as Pick's disease; subcortical dementias (such as
Huntington or progressive supranuclear palsy); focal cortical
atrophy syndromes (such as primary aphasia); metabolic-toxic
dementias (such as chronic hypothyroidism or B12 deficiency); and
dementias caused by infections (such as syphilis or chronic
meningitis).
[0231] Alzheimer's disease is characterized by extracellular
deposits of .beta.-amyloid protein (.beta.-AP) in senile plaques
and cerebral vessels. .beta.-AP is a peptide fragment of 39 to 42
amino acids derived from an amyloid protein precursor (APP). At
least three isoforms of APP are known (695, 751, and 770 amino
acids). Alternative splicing of mRNA generates the isoforms; normal
processing affects a portion of the .beta.-AP sequence, thereby
preventing the generation of .beta.-AP. It is believed that
abnormal protein processing by the proteasome contributes to the
abundance of .beta.-AP in the Alzheimer brain. The APP-processing
enzyme in rats contains about ten different subunits (22 kDa-32
kDa). The 25 kDa subunit has an N-terminal sequence of
X-Gln-Asn-Pro-Met-X-Thr-Gly-Thr-Ser, which is identical to the
.beta.-subunit of human macropain (Kojima, S., et al., Fed. Eur.
Biochem. Soc. 304:57-60 (1992)). The APP-processing enzyme cleaves
at the Gln15-Lys16 bond; in the presence of calcium ion, the enzyme
also cleaves at the Met-1-Asp1 bond, and the Asp1-Ala2 bonds to
release the extracellular domain of .beta.-AP.
[0232] In yet another embodiment of the present invention, the
liposomal compositions can be used as a treatment for Alzheimer's
disease, comprising administering to a subject an effective amount
of peptide epoxyketone-containing liposomal compositions disclosed
herein. In such cases, the liposomal compositions reduce the rate
of .beta.-AP processing, reduce the rate of .beta.-AP plaque
formation, reduce the rate of .beta.-AP generation, and reduce the
clinical signs of Alzheimer's disease.
[0233] The proteasome degrades many proteins in maturing
reticulocytes and growing fibroblasts. In cells deprived of insulin
or serum, the rate of proteolysis nearly doubles Inhibiting the
proteasome reduces proteolysis, thereby reducing both muscle
protein loss and the nitrogenous load on kidneys or liver.
Liposomal compositions comprising peptide epoxyketone compounds as
provided herein are useful for treating conditions such as cancer,
chronic infectious diseases, fever, muscle disuse (atrophy) and
denervation, nerve injury, fasting, renal failure associated with
acidosis, and hepatic failure. See, e.g., U.S. Pat. No. 5,340,736.
Methods of treatment include: reducing the rate of muscle protein
degradation in a cell; reducing the rate of intracellular protein
degradation; reducing the rate of degradation of p53 protein in a
cell; and inhibiting the growth of p53-related cancers. Each of
these methods includes contacting a cell (in vivo or in vitro,
e.g., a muscle in a subject) with an effective amount of a
pharmaceutical liposomal composition disclosed herein.
[0234] Other embodiments of the present invention relate to methods
for treating cachexia and muscle-wasting diseases, cancers, chronic
infectious diseases, fever, muscle disuse (atrophy) and
denervation, nerve injury, fasting, renal failure associated with
acidosis, diabetes, and hepatic failure. Embodiments of the
invention encompass methods for: reducing the rate of muscle
protein degradation in a cell; reducing the rate of intracellular
protein degradation; reducing the rate of degradation of p53
protein in a cell; and inhibiting the growth of p53-related
cancers.
[0235] Fibrosis is the excessive and persistent formation of scar
tissue resulting from the hyperproliferative growth of fibroblasts
and is associated with activation of the TGF-.beta. signaling
pathway. Fibrosis involves extensive deposition of extracellular
matrix and can occur within virtually any tissue or across several
different tissues. Normally, the level of intracellular signaling
protein (Smad) that activate transcription of target genes upon
TGF-.beta. stimulation is regulated by proteasome activity.
However, accelerated degradation of the TGF-.beta. signaling
components has been observed in cancers and other
hyperproliferative conditions.
[0236] Another protein processed by the proteasome is NF-.kappa.B,
a member of the Rel protein family. The Rel family of
transcriptional activator proteins can be divided into two groups.
The first group requires proteolytic processing, and includes p50
(NF-.kappa.B1, 105 kDa) and p52 (NF-.kappa.2, 100 kDa). The second
group does not require proteolytic processing, and includes p65
(RelA, Rel (c-Rel), and RelB). Both homo- and heterodimers can be
formed by Rel family members; NF-.kappa.B, for example, is a
p50-p65 heterodimer. After phosphorylation and ubiquitination of
I.kappa.B and p105, the two proteins are degraded and processed,
respectively, to produce active NF-.kappa.B which translocates from
the cytoplasm to the nucleus. Ubiquitinated p105 is also processed
by purified proteasomes (Palombella, et al., Cell 78:773-785
(1994)). Active NF-.kappa.B forms a stereospecific enhancer complex
with other transcriptional activators and, e.g., HMG I(Y), inducing
selective expression of a particular gene.
[0237] NF-.kappa.B regulates genes involved in the immune and
inflammatory response, and mitotic events. For example, NF-.kappa.B
is required for the expression of the immunoglobulin light chain
.kappa. gene, the IL-2 receptor .alpha.-chain gene, the class I
major histocompatibility complex gene, and a number of cytokine
genes encoding, for example, IL-2, IL-6, granulocyte
colony-stimulating factor, and IFN-.beta. (Palombella, et al., Cell
78:773-785 (1994)). Some embodiments include methods of affecting
the level of expression of IL-2, MHC-I, IL-6, TNF.alpha.,
IFN-.beta., or any of the other previously-mentioned proteins, each
method including administering to a subject a therapeutically
effective amount of a liposomal composition comprising a peptide
epoxyketone compound as disclosed herein. Complexes including p50
are rapid mediators of acute inflammatory and immune responses
(Thanos, D. and Maniatis, T., Cell 80:529-532 (1995)).
[0238] NF-.kappa.B also participates in the expression of the cell
adhesion genes that encode E-selectin, P-selectin, ICAM, and VCAM-1
(Collins, T., Lab. Invest. 68:499-508 (1993)). In some embodiments,
a method for inhibiting cell adhesion (e.g., cell adhesion mediated
by E-selectin, P-selectin, ICAM, or VCAM-1) is provided, including
contacting a cell with (or administering to a subject) an effective
amount of a liposomal composition comprising a peptide expoxyketone
compound disclosed herein.
[0239] Certain embodiments of the present invention relate to a
method for treating hyperproliferative conditions such as diabetic
retinopathy, macular degeneration, diabetic nephropathy,
glomerulosclerosis, IgA nephropathy, cirrhosis, biliary atresia,
congestive heart failure, scleroderma, radiation-induced fibrosis,
and lung fibrosis (idiopathic pulmonary fibrosis, collagen vascular
disease, sarcoidosis, interstitial lung diseases, and extrinsic
lung disorders). The treatment of burn victims often is hampered by
fibrosis; thus, an additional embodiment of the invention is the
topical or systemic administration of the peptide
epoxyketone-containing liposomal composition for burn treatment.
Wound closure following surgery often is associated with
disfiguring scars, which can be prevented by inhibition of
fibrosis. Thus, in certain embodiments, the invention relates to a
method for prevention or reduction of scarring.
[0240] Ischemia and reperfusion injury results in hypoxia, a
condition in which there is a deficiency of oxygen reaching the
tissues of the body. This condition causes increased degradation of
I.kappa.-B.alpha., thereby resulting in the activation of
NF-.kappa.B. It has been demonstrated that the severity of injury
resulting in hypoxia can be reduced with the administration of a
proteasome inhibitor. Thus, provided herein is a method of treating
an ischemic condition or reperfusion injury comprising
administering to a subject in need of such treatment a
therapeutically effective amount of a liposomal composition
comprising a peptide epoxyketone compound as disclosed herein.
[0241] Certain embodiments of the present invention relate to a
method of treating ischemia and reperfusion injury, which are
associated with hypoxia, a deficiency of oxygen reaching the
tissues of the body. Examples of such injuries or conditions
include, but are not limited to, acute coronary syndrome
(vulnerable plaques), arterial occlusive disease (cardiac,
cerebral, peripheral arterial and vascular occlusions),
atherosclerosis (coronary sclerosis, coronary artery disease),
infarctions, heart failure, pancreatitis, myocardial hypertrophy,
stenosis, and restenosis.
[0242] NF-.kappa.B also binds specifically to the
HIV-enhancer/promoter. When compared to the Nef of mac239, the HIV
regulatory protein Nef of pbj14 differs by two amino acids in the
region which controls protein kinase binding. It is believed that
the protein kinase signals the phosphorylation of I.kappa.B,
triggering I.kappa.B degradation through the ubiquitin-proteasome
pathway. After degradation, NF-.kappa.B is released into the
nucleus, thus enhancing the transcription of HIV (Cohen, J.,
Science, 267:960 (1995)). Provided herein is a method for
inhibiting or reducing HIV infection in a subject, and a method for
decreasing the level of viral gene expression, each method
including administering to the subject a therapeutically effective
amount of a liposomal composition comprising a peptide epoxyketone
compound as disclosed herein.
[0243] Two further embodiments of the present invention are a
method for inhibiting or reducing HIV infection in a subject, and a
method for decreasing the level of viral gene expression.
[0244] Viral infections contribute to the pathology of many
diseases. Heart conditions such as ongoing myocarditis and dilated
cardiomyopathy have been linked to the coxsackievirus B3. In a
comparative whole-genome microarray analyses of infected mouse
hearts, specific proteasome subunits were uniformly up-regulated in
hearts of mice that developed chronic myocarditis (Szalay, et al.,
Am J Pathol 168:1542-52 (2006)). Some viruses utilize the
ubiquitin-proteasome system in the viral entry step where the virus
is released from the endosome into the cytosol. The mouse hepatitis
virus (MHV) belongs to the Coronaviridae family, which also
includes the severe acute respiratory syndrome (SARS) coronvirus.
Yu and Lai (J Virol 79:644-648 (2005)) demonstrated that treatment
of cells infected with MHV with a proteasome inhibitor resulted in
a decrease in viral replication, correlating with reduced viral
titer as compared to that of untreated cells. The human hepatitis B
virus (HBV), a member of the Hepadnaviridae virus family, likewise
requires virally encoded envelop proteins to propagate Inhibiting
the proteasome degradation pathway causes a significant reduction
in the amount of secreted envelope proteins (Simsek, et al., J
Virol 79:12914-12920 (2005)). In addition to HBV, other hepatitis
viruses (A, C, D and E) may also utilize the ubiquitin-proteasome
degradation pathway for secretion, morphogenesis and pathogenesis.
Accordingly, in certain embodiments, a method for treating viral
infection, such as SARS or hepatitis A, B, C, D and E, is provided
comprising contacting a cell with (or administering to a subject)
an effective amount of a liposomal composition comprising a peptide
epoxyketone compound as disclosed herein.
[0245] Overproduction of lipopolysaccharide (LPS)-induced cytokines
such as TNF.alpha. is considered to be central to the processes
associated with septic shock. Furthermore, it is generally accepted
that the first step in the activation of cells by LPS is the
binding of LPS to specific membrane receptors. The .alpha.- and
.beta.-subunits of the 20S proteasome complex have been identified
as LPS-binding proteins, suggesting that the LPS-induced signal
transduction may be an important therapeutic target in the
treatment or prevention of sepsis (Qureshi, N., et al., J. Immun.
171: 1515-1525 (2003)).
[0246] In certain embodiments, compounds of the present invention
can be used for the inhibition of TNF.alpha. to prevent and/or
treat septic shock.
[0247] Intracellular proteolysis generates small peptides for
presentation to T-lymphocytes to induce MHC class I-mediated immune
responses. The immune system screens for autologous cells that are
virally infected or have undergone oncogenic transformation. One
embodiment is a method for inhibiting antigen presentation in a
cell, including exposing the cell to a liposomal composition
comprising a peptide epoxyketone compound as described herein. A
further embodiment is a method for suppressing the immune system of
a subject (e g., inhibiting transplant rejection, allergy, asthma),
including administering to the subject a therapeutically effective
amount of a liposomal composition comprising a peptide epoxyketone
compound as described herein. Liposomal compositions provided
herein can also be used to treat autoimmune diseases such as lupus,
rheumatoid arthritis, multiple sclerosis, and inflammatory bowel
diseases such as ulcerative colitis and Crohn's disease.
[0248] Another embodiment is a method for altering the repertoire
of antigenic peptides produced by the proteasome or other Ntn with
multicatalytic activity. For example, if the PGPH activity of 20S
proteasome is selectively inhibited, a different set of antigenic
peptides will be produced by the proteasome and presented in MHC
molecules on the surfaces of cells than would be produced and
presented either without any enzyme inhibition, or with, for
example, selective inhibition of chymotrypsin-like activity of the
proteasome.
[0249] An additional embodiment of the present invention is a
method for inhibiting antigen presentation in a cell. In such
method, the liposomal composition is used to treat immune-related
conditions such as allergy, asthma, organ/tissue rejection
(graft-versus-host disease), and auto-immune diseases, including,
but not limited to, lupus, rheumatoid arthritis, psoriasis,
multiple sclerosis, and inflammatory bowel diseases (such as
ulcerative colitis and Crohn's disease). Thus, a further embodiment
is a method for suppressing the immune system of a subject (e g.,
inhibiting transplant rejection, allergies, auto-immune diseases,
and asthma), including administering to the subject an effective
amount of a liposomal composition comprising a peptide epoxyketone
compound described herein.
[0250] Certain proteasome inhibitors block both degradation and
processing of ubiquitinated NF-.kappa.B in vitro and in vivo.
Proteasome inhibitors also block I.kappa.B-a degradation and
NF-.kappa.B activation (Palombella, et al. Cell 78:773-785 (1994);
and Traenckner, et al., EMBO J. 13:5433-5441 (1994)). In some
embodiments, a method for inhibiting I.kappa.B-a degradation is
provided, including contacting the cell with a liposomal
composition comprising a peptide epoxyketone compound as described
herein. A further embodiment is a method for reducing the cellular
content of NF-.kappa.B in a cell, muscle, organ, or subject,
including contacting the cell, muscle, organ, or subject with a
liposomal composition comprising a peptide epoxyketone compound as
described herein.
[0251] Other eukaryotic transcription factors that require
proteolytic processing include the general transcription factor
TFIIA, herpes simplex virus VP16 accessory protein (host cell
factor), virus-inducible IFN regulatory factor 2 protein, and the
membrane-bound sterol regulatory element-binding protein 1.
[0252] Further provided herein are methods for affecting
cyclin-dependent eukaryotic cell cycles, including exposing a cell
(in vitro or in vivo) to a liposomal composition comprising a
peptide epoxyketone compound disclosed herein. Cyclins are proteins
involved in cell cycle control. The proteasome participates in the
degradation of cyclins. Examples of cyclins include mitotic
cyclins, G1 cyclins, and cyclin B. Degradation of cyclins enables a
cell to exit one cell cycle stage (e.g., mitosis) and enter another
(e.g., division). It is believed all cyclins are associated with
p34cdc2 protein kinase or related kinases. The proteolysis
targeting signal is localized to amino acids 42-RAALGNISEN-50
(destruction box). There is evidence that cyclin is converted to a
form vulnerable to a ubiquitin ligase or that a cyclin-specific
ligase is activated during mitosis (Ciechanover, A., Cell, 79:13-21
(1994)). Inhibition of the proteasome inhibits cyclin degradation,
and therefore inhibits cell proliferation, for example, in
cyclin-related cancers (Kumatori, et al., Proc. Natl. Acad. Sci.
USA 87:7071-7075 (1990)). Provided herein is a method for treating
a proliferative disease in a subject (e.g., cancer, psoriasis, or
restenosis), including administering to the subject a
therapeutically effective amount of a composition disclosed herein.
Also provided herein is a method for treating cyclin-related
inflammation in a subject, including administering to a subject a
therapeutically effective amount of a liposomal composition
comprising a peptide epoxyketone compound as described herein.
[0253] Additional embodiments include methods for affecting the
proteasome-dependent regulation of oncoproteins and methods of
treating or inhibiting cancer growth, each method including
exposing a cell (in vivo, e.g., in a subject, or in vitro) to a
liposomal composition comprising a peptide epoxyketone compound as
disclosed herein. HPV-16 and HPV-18-derived E6 proteins stimulate
ATP- and ubiquitin-dependent conjugation and degradation of p53 in
crude reticulocyte lysates. The recessive oncogene p53 has been
shown to accumulate at the nonpermissive temperature in a cell line
with a mutated thermolabile E1. Elevated levels of p53 may lead to
apoptosis. Examples of proto-oncoproteins degraded by the ubiquitin
system include c-Mos, c-Fos, and c-Jun. One embodiment is a method
for treating p53-related apoptosis, including administering to a
subject a therapeutically effective amount of a liposomal
composition comprising a peptide epoxyketone compound as disclosed
herein.
[0254] One embodiment of the invention is a method for inhibiting
I.kappa.B-.alpha. degradation, including contacting the cell with
the liposomal composition. A further embodiment is a method for
reducing the cellular content of NF-.kappa.B in a cell, muscle,
organ, or subject, including contacting the cell, muscle, organ, or
subject with a liposomal composition comprising a peptide
epoxyketone compound.
[0255] A further embodiment of the invention is a method for
treating a proliferative disease in a subject (e.g., cancer,
psoriasis, or restenosis), including administering to the subject
an effective amount of the liposomal composition. The invention
also encompasses a method for treating cyclin-related inflammation
in a subject.
[0256] Another embodiment of the present invention is a method for
treating p53-related apoptosis.
[0257] The proteasome of these parasites is considered to be
involved primarily in cell differentiation and replication
activities (Paugam, et al., Trends Parasitol. 19(2): 55-59(2003)).
Furthermore, entamoeba species have been shown to lose encystation
capacity when exposed to proteasome inhibitors (Gonzales, et al.,
Arch. Med. Res. 28, Spec No: 139-140 (1997)). Other compounds
useful as proteasome inhibitors in the treatment of parasitic
diseases are described in WO 98/10779.
[0258] In certain embodiments, the disclosed compositions inhibit
proteasome activity irreversibly in a parasite. Such irreversible
inhibition has been shown to induce shutdown in enzyme activity
without recovery in red blood cells and white blood cells. In
certain such embodiments, the long half-life of blood cells may
provide prolonged protection with regard to therapy against
recurring exposures to parasites. In certain embodiments, the long
half-life of blood cells may provide prolonged protection with
regard to chemoprophylaxis against future infection.
[0259] In a certain embodiments, the invention's liposomal
compositions are useful for the treatment of a parasitic infection,
such as infections in humans caused by a protozoan parasite
selected from Plasmodium sps. (including P. falciparum, P. vivax,
P. malariae, and P. ovale, which cause malaria), Trypanosoma sps.
(including T. cruzi, which causes Chagas' disease, and T. brucei
which causes African sleeping sickness), Leishmania sps. (including
L. amazonensis, L. donovani, L. infantum, L. mexicana, etc.),
Pneumocystis carinii (a protozoan known to cause pneumonia in AIDS
and other immunosuppressed patients), Toxoplasma gondii, Entamoeba
histolytica, Entamoeba invadens, and Giardia lamblia. In certain
embodiments, the disclosed liposomal compositions comprising
peptide epoxyketone compounds are useful for the treatment of
parasitic infections in animals and livestock caused by a protozoan
parasite selected from Plasmodium hermani, Cryptosporidium sps.,
Echinococcus granulosus, Eimeria tenella, Sarcocystis neurona, and
Neurospora crassa.
[0260] Prokaryotes have what is equivalent to the eukaryote 20S
proteasome particle. Albeit, the subunit composition of the
prokaryote 20S particle is simpler than that of eukaryotes, it has
the ability to hydrolyze peptide bonds in a similar manner. For
example, the nucleophilic attack on the peptide bond occurs through
the threonine residue on the N-terminus of the .beta.-subunits. In
some embodiments, a method of treating prokaryotic infections is
provided, comprising administering to a subject a therapeutically
effective amount of a liposomal composition comprising a peptide
epoxyketone compound as disclosed herein. Prokaryotic infections
may include diseases caused by either mycobacteria (such as
tuberculosis, leprosy or Buruli Ulcer) or archaebacteria.
[0261] It has also been demonstrated that inhibitors that bind to
the 20S proteasome stimulate bone formation in bone organ cultures.
Furthermore, when such inhibitors have been administered
systemically to mice, certain proteasome inhibitors increased bone
volume and bone formation rates over 70% (Garrett, I. R., et al.,
J. Clin. Invest. 111: 1771-1782 (2003)), therefore suggesting that
the ubiquitin-proteasome machinery regulates osteoblast
differentiation and bone formation. Therefore, the disclosed
liposomal compositions comprising peptide epoxyketone compounds may
be useful in the treatment and/or prevention of diseases associated
with bone loss, such as osteoporosis.
[0262] Provided herein is a method for treating a disease or
condition selected from cancer, autoimmune disease, graft or
transplant-related condition, neurodegenerative disease,
fibrotic-associated condition, ischemic-related conditions,
infection (viral, parasitic or prokaryotic) and diseases associated
with bone loss, comprising administering a therapeutically
effective amount of a liposomal composition comprising a peptide
epoxyketone compound as provided herein.
[0263] Bone tissue is an excellent source for factors which have
the capacity for stimulating bone cells. Thus, extracts of bovine
bone tissue contain not only structural proteins that are
responsible for maintaining the structural integrity of bone, but
also biologically active bone growth factors that can stimulate
bone cells to proliferate. Among these latter factors is a recently
described family of proteins called bone morphogenetic proteins
(BMPs). All of these growth factors have effects on other types of
cells, as well as on bone cells (see e.g., Hardy, M. H., et al.,
Trans Genet 8:55-61 (1992), which describes evidence that bone
morphogenetic proteins (BMPs) are differentially expressed in hair
follicles during development; BMP-2 expression in mature follicles
also occurs during maturation and after the period of cell
proliferation (Hardy, M. H., et al., (1992, supra); Harris, S. E.,
et al., J Bone Miner Res 9:855-863 (1994), which describes the
effects of TGF-.beta. on expression of BMP-2 and other substances
in bone cells). Thus, liposomal compositions comprising peptide
epoxyketone compounds as provided herein may also be useful for
hair follicle growth stimulation.
[0264] In one embodiment of the present invention, the liposomal
compositions can be useful in the treatment and/or prevention of
diseases associated with bone loss, such as osteoporosis.
[0265] Finally, the disclosed liposomal compositions comprising
peptide epoxyketone compounds are also useful as diagnostic agents
(e.g., in diagnostic kits or for use in clinical laboratories) for
screening for proteins (e.g., enzymes, transcription factors)
processed by Ntn hydrolases, including the proteasome. The
disclosed liposomal compositions are also useful as research
reagents for specifically binding the X/MB1 subunit or
.alpha.-chain and inhibiting the proteolytic activities associated
with it. For example, the activity of (and specific inhibitors of)
other subunits of the proteasome can be determined.
[0266] Actual dosage levels of peptide epoxyketone compounds in
pharmaceutical compositions of this invention can be varied so as
to obtain an amount of the peptide epoxyketone compound that is
effective to achieve the desired therapeutic response for a
particular subject, composition, and mode of administration,
without being toxic to the subject.
[0267] The concentration of peptide epoxyketone compound in a
pharmaceutically acceptable mixture will vary depending on several
factors, including dosage of the compound to be administered,
pharmacokinetic characteristics of the compound(s) employed, and
route of administration. In general, the liposomal compositions of
this invention can be provided in an aqueous solution for
parenteral administration. Typical dose ranges are from about 0.01
mg/kg to about 50 mg/kg of body weight per day of peptide
epoxyketone compound. Exemplary dose ranges include between about
10 mg/m.sup.2 and about 150 mg/m.sup.2(mg/m.sup.2, milligrams per
square meter of body surface of the subject to whom the liposomal
composition is administered) of peptide epoxyketone compound,
preferably between about 15 mg/m.sup.2 and about 70 mg/m.sup.2,
more preferably between about 15 mg/m.sup.2 and about 56
mg/m.sup.2. Administration of the liposomal compositions of the
present invention is typically intravenously, once or twice weekly,
and can be administered in single or divided doses. Each divided
dose may contain the same or different peptide epoxyketone
compounds. The dosage will be an effective amount depending on
several factors including the overall health of a patient, and the
composition and route of administration of the selected peptide
epoxyketone compound(s). Dosage forms (also called unit doses) of
liposomal compositions of the present invention are typically
single-use vials comprising between about 20 mg and about 300 mg of
peptide epoxyketone compound, preferably between about 30 mg and
about 140 mg, more preferably between about 30 mg and about 112
mg.
[0268] Another aspect of the present invention provides a
combination treatment wherein one or more other therapeutic agents
are administered with the peptide epoxyketone-containing liposomal
composition. Such combination treatment can be achieved by
simultaneous, sequential, or separate dosing of the individual
components of the treatment.
[0269] In certain embodiments of the present invention, a peptide
epoxyketone-containing liposomal composition described herein is
used as part of a combination treatment that includes one or more
other proteasome inhibitor(s).
[0270] In other embodiments, a liposomal composition of the
invention is part of a combination treatment that includes a
chemotherapeutic. Suitable chemotherapeutics may include natural
products such as vinca alkaloids (i.e., vinblastine, vincristine,
and vinorelbine), paclitaxel, epidipodophyllotoxins (i.e.,
etoposide, teniposide), antibiotics (dactinomycin (actinomycin D),
daunorubicin, doxorubicin, and idarubicin), anthracyclines,
mitoxantrone, bleomycins, plicamycin (mithramycin), and mitomycin,
enzymes (L-asparaginase, which systemically metabolizes
L-asparagine and deprives cells that do not have the capacity to
synthesize their own asparagine); antiplatelet agents;
antiproliferative/antimitotic alkylating agents such as nitrogen
mustards (mechlorethamine, cyclophosphamide, and analogs,
melphalan, chlorambucil), ethylenimines and methylmelamines
(hexamethylmelamine and thiotepa), alkyl sulfonates (busulfan),
nitrosoureas (carmustine (BCNU) and analogs, streptozocin),
trazenes-dacarbazinine (DTIC); antiproliferative/antimitotic
antimetabolites such as folic acid analogs (methotrexate),
pyrimidine analogs (fluorouracil, floxuridine, and cytarabine),
purine analogs and related inhibitors (mercaptopurine, thioguanine,
pentostatin, and 2-chlorodeoxyadenosine); aromatase inhibitors
(anastrozole, exemestane, and letrozole); and platinum coordination
complexes (cisplatin, carboplatin), procarbazine, hydroxyurea,
mitotane, aminoglutethimide; histone deacetylase (HDAC) inhibitors
(trichostatin, sodium butyrate, apicidan, suberoyl anilide
hydroamic acid); hormones (i.e., estrogen) and hormone agonists
such as leutinizing hormone releasing hormone (LHRH) agonists
(goserelin, leuprolide, and triptorelin). Other chemotherapeutic
agents may include lenalidomide, mechlorethamine, camptothecin,
ifosfamide, tamoxifen, raloxifene, gemcitabine, navelbine, or any
analog or derivative variant of the foregoing.
[0271] In certain embodiments, a liposomal composition comprising a
peptide epoxyketone compound as described herein is conjointly
administered with one or more histone deacetylase (HDAC) inhibitors
(e.g., trichostatin, sodium butyrate, apicidan, suberoyl anilide
hydroamic acid ("SAHA" (Vorinostat)), trichostatin A, depsipeptide,
apicidin, A-161906, scriptaid, PXD-101, CHAP, butyric acid,
depudecin, oxamflatin, phenylbutyrate, valproic acid, MS275
(N-(2-Aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzam-
ide), LAQ824/LBH589, CI994, MGCD0103, ACY-1215, Panobinostat; e.g.,
SAHA, ACY-1215, Panobinostat).
[0272] In certain embodiments, liposomal compositions comprising
peptide epoxyketone compounds as described herein are conjointly
administered with one or more DNA binding/Cytotoxic agents (e.g.,
Zalypsis).
[0273] In certain embodiments, a liposomal composition comprising a
peptide epoxyketone compound as described herein is conjointly
administered with one or more taxanes (e.g., docetaxel, and/or
paclitaxel).
[0274] In certain embodiments, liposomal compositions comprising
peptide epoxyketone compounds as described herein are conjointly
administered with dexamethasone. For example, treatment may include
pre-medicating with dexamethasone 4 mg orally or intravenously
prior to all doses of liposomal compositions comprising peptide
epoxyketone compounds during a first treatment cycle and prior to
all doses of liposomal compositions comprising peptide epoxyketone
compounds during a treatment cycle with dose escalation to reduce
the incidence and severity of infusion reactions. Such
dexamethasone premedication (e.g., 4 mg orally or intravenously) is
administered if such reactions develop or reappear during
subsequent treatment cycles.
[0275] In some embodiments, the present invention relates to a
method of treating cancer (e.g., multiple myeloma or solid tumor)
in a subject in need of treatment. The method typically comprises
administering a therapeutically effective amount of a
pharmaceutical liposomal composition of the present invention
(e.g., comprising carfilzomib), and may further comprise
simultaneous, sequential, or separate administration of a
therapeutically effective amount of a chemotherapeutic agent.
[0276] In certain embodiments of the present invention, a liposomal
composition described herein is used in a combination treatment
that includes a cytokine Cytokines include, but are not limited to,
Interferon-.gamma., Interferon-.alpha., and Interferon-.beta.;
Interleukins 1-8, 10, and 12; Granulocyte Monocyte
Colony-Stimulating Factor (GM-CSF); TNF-.alpha. and TNF-.beta.; and
TGF-.beta..
[0277] Embodiments of the present invention include combination
treatments incorporating a liposomal composition described herein
and a steroid. Suitable steroids may include, but are not limited
to, 21-acetoxypregnenolone, alclometasone, algestone, amcinonide,
beclomethasone, betamethasone, budesonide, chloroprednisone,
clobetasol, clocortolone, cloprednol, corticosterone, cortisone,
cortivazol, deflazacort, desonide, desoximetasone, dexamethasone,
diflorasone, diflucortolone, difuprednate, enoxolone, fluazacort,
flucloronide, flumethasone, flunisolide, fluocinolone acetonide,
fluocinonide, fluocortin butyl, fluocortolone, fluorometholone,
fluperolone acetate, fluprednidene acetate, fluprednisolone,
flurandrenolide, fluticasone propionate, formocortal, halcinonide,
halobetasol propionate, halometasone, hydrocortisone, loteprednol
etabonate, mazipredone, medrysone, meprednisone,
methylprednisolone, mometasone furoate, paramethasone,
prednicarbate, prednisolone, prednisolone 25-diethylaminoacetate,
prednisolone sodium phosphate, prednisone, prednival, prednylidene,
rimexolone, tixocortol, triamcinolone, triamcinolone acetonide,
triamcinolone benetonide, triamcinolone hexacetonide, and salts
and/or derivatives thereof.
[0278] In certain embodiments of the present invention, a liposomal
composition described herein is part of a combination treatment
that includes an immunotherapeutic agent. Suitable
immunotherapeutic agents may include, but are not limited to, MDR
modulators (verapamil, valspordar, biricodar, tariquidar,
laniquidar), cyclosporine, thalidomide, and monoclonal antibodies).
The monoclonal antibodies can be either naked or conjugated such as
rituximab, tositumomab, alemtuzumab, epratuzumab, ibritumomab
tiuxetan, gemtuzumab ozogamicin, bevacizumab, cetuximab, erlotinib,
and trastuzumab. In certain embodiments, a liposomal composition of
the present disclosure described herein is conjointly administered
with lenalidomide (REVLIMID.RTM., Celgene Corporation, Summit,
N.J.).
[0279] In some embodiments, a liposomal composition comprising a
peptide epoxyketone compound as described herein is conjointly
administered with one or more topoisomerase inhibitors (e.g.,
irinotecan, topotecan, camptothecin, lamellarin D, etoposide).
[0280] In some embodiments, a liposomal composition comprising a
peptide epoxyketone compound as described herein is conjointly
administered with one or more m-TOR inhibitors (e.g., CCI-779,
AP23573 and RAD-001).
[0281] In some embodiments, a liposomal composition comprising a
peptide epoxyketone compound as described herein is conjointly
administered with one or more protein kinase inhibitor (e.g.,
sorafenib, imatinib, dasatinib, sunitinib, pazopanib, and
nilotinib; e.g., sorafenib).
[0282] In some embodiments, a liposomal composition comprising a
peptide epoxyketone compound as described herein is conjointly
administered with one or more CDK Inhibitors (e.g.,
Dinaciclib).
[0283] In some embodiments, a liposomal composition comprising a
peptide epoxyketone compound as described herein is conjointly
administered with one or more KSP(Eg5) Inhibitors (e.g., Array
520).
[0284] In some embodiments, a liposomal composition comprising a
peptide epoxyketone compound as described herein is conjointly
administered with one or more PI13 delta Inhibitors (e.g., GS-1101
PI3K).
[0285] In some embodiments, a liposomal composition comprising a
peptide epoxyketone compound as described herein is conjointly
administered with one or more Dual Inhibitor: PI3K delta and gamma
Inhibitors (e.g., CAL-130).
[0286] In some embodiments, a liposomal composition comprising a
peptide epoxyketone compound as described herein is conjointly
administered with one or more multi-kinase Inhibitors (e.g.,
TG02).
[0287] In some embodiments, a liposomal composition comprising a
peptide epoxyketone compound as described herein is conjointly
administered with one or more PI3K delta Inhibitors (e.g.,
TGR-1202).
[0288] In some embodiments, a liposomal composition comprising a
peptide epoxyketone compound as described herein is conjointly
administered with:
[0289] (i) one or more of the following:
[0290] one or more second chemotherapeutic agents (e.g., one or
more HDAC inhibitors (e.g., SAHA, ACY-1215, Panobinostat); one or
more nitrogen mustards (e.g., melphalan); one or more DNA
binding/cytotoxic agents (e.g., Zylapsis); one or more taxanes
(e.g., docetaxel); one or more antibiotics (e.g., dactinomycin
(actinomycin D), daunorubicin, doxorubicin and idarubicin; e.g.,
doxorubicin);
[0291] one or more other peptide epoxyketone compound (e.g.,
another compound of formulae (I)-(V));
[0292] one or more cytokines;
[0293] one or more immunotherapeutic agents (e.g.,
REVLIMID.RTM.);
[0294] one or more topoisomerase inhibitors;
[0295] one or more m-TOR inhibitors;
[0296] one or more protein kinase inhibitor (e.g., sorafenib);
[0297] one or more CDK Inhibitors (e.g., Dinaciclib);
[0298] one or more KSP(Eg5) Inhibitors (e.g., Array 520);
[0299] one or more PI13 delta Inhibitors (e.g., GS-1101 PI3K);
[0300] one or more Dual Inhibitor: PI3K delta and gamma Inhibitors
(e.g., CAL-130);
[0301] one or more multi-kinase Inhibitors (e.g., TG02);
[0302] one or more PI3K delta Inhibitors (e.g., TGR-1202);
[0303] and
[0304] (ii) one or more steroids (e.g., dexamethasone).
[0305] In certain embodiments, a liposomal composition comprising a
peptide epoxyketone compound as described herein is conjointly
administered with:
[0306] (i) one of the following:
[0307] one or more second chemotherapeutic agents (e.g., one or
more HDAC inhibitors, (e.g., SAHA, ACY-1215, Panobinostat); one or
more nitrogen mustards (e.g., melphalan); one or more DNA
binding/cytotoxic agents, (e.g., Zylapsis); one or more taxanes
(e.g., docetaxel); one or more antibiotics (e.g., dactinomycin
(actinomycin D), daunorubicin, doxorubicin and idarubicin; e.g.,
doxorubicin);
[0308] one or more other peptide epoxyketone compound (e.g.,
another compound of formulae (I)-(V));
[0309] one or more cytokines;
[0310] one or more immunotherapeutic agents (e.g.,
REVLIMID.RTM.);
[0311] one or more topoisomerase inhibitors;
[0312] one or more m-TOR inhibitors;
[0313] one or more protein kinase inhibitor (e.g., sorafenib);
[0314] one or more CDK Inhibitors (e.g., Dinaciclib);
[0315] one or more KSP(Eg5) Inhibitors (e.g., Array 520);
[0316] one or more PI13 delta Inhibitors (e.g., GS-1101 PI3K);
[0317] one or more Dual Inhibitor: PI3K delta and gamma Inhibitors
(e.g., CAL-130);
[0318] one or more multi-kinase Inhibitors (e.g., TG02);
[0319] one or more PI3K delta Inhibitors (e.g., TGR-1202);
[0320] and
[0321] (ii) dexamethasone.
[0322] Experiments performed in support of the present invention
demonstrated that liposomal compositions of the present invention
provided increased maximum tolerated dose (MTD) relative to a
non-liposomal composition comprising peptide epoxyketone compound.
For example, in mice, a first liposomal composition resulted in a
2.5-fold increase in the MTD, and a second liposomal composition
resulted in a 50% increase. In rats, both liposomal compositions
resulted in increases in tolerability (Example 4). Biodistribution,
as measured by proteasome inhibition in blood and tissues, was
similar across the various compositions (Example 5, FIG. 2A-2D;
Example 8, FIG. 3A-3D; Example 11, FIG. 5A-5D). Further, the
liposomal peptide epoxyketone compound compositions of the present
invention provided about 3 to 5 and 7-fold increased exposure (AUC)
in mice and rats, respectively, compared to a non-liposomal
composition comprising peptide epoxyketone compound. This increased
exposure was the result of a decrease in plasma clearance (Example
6).
[0323] Further, liposomal compositions comprising a peptide
epoxyketone compound entrapped in the liposomes' aqueous core
demonstrated enhanced tolerability by increasing the maximum
tolerated dose (MTD) of carfilzomib in mice by 50%, from 10 mg/kg
to 15 mg/kg as compared to the injectable, non-liposomal, SBE-B-CD
composition. These results indicate that liposomal compositions
comprising a peptide epoxyketone compound entrapped in the
liposomes' aqueous core release carfilzomib over a longer period of
time with a lower maximum plasma concentration (Cmax) relative to
the injectable, non-liposomal, SBE-B-CD composition.
[0324] The liposomal compositions comprising liposomes having a
peptide epoxyketone compound entrapped in their aqueous core also
resulted in delayed proteasome recovery at 24 hours in some mouse
tissues whereas the current drug product (i.e., injectable,
non-liposomal, CFZ SBE-B-CD) resulted in recovery from proteasome
inhibition by 24 hours post-dose (Example 8, FIG. 3A, FIG. 3B, FIG.
3C, FIG. 3D). These results support that the liposomal compositions
provide long-term exposure of peptide epoxyketone compounds.
[0325] When delivered in the non-liposomal CFZ SBE-B-CD
composition, the plasma concentration of carfilzomib declined
rapidly and was not detectable after 1 hour post-dose (Example 11,
FIG. 6). When carfilzomib was delivered as liposomal compositions
comprising a peptide epoxyketone compound entrapped in the
liposomes, systemic exposure was extended with detectable total
drug (both encapsulated and released) for up to 24 hours post-dose
(Example 11, FIG. 6). These data demonstrate that the liposomal
compositions comprising a peptide epoxyketone compound entrapped in
the liposomes resulted in greater exposure and longer circulation
relative to a non-liposomal, peptide epoxyketone compound
composition.
[0326] In addition, when delivered in the non-liposomal CFZ
SBE-B-CD composition, the plasma concentration of carfilzomib
declined rapidly and was not detectable after 1 hour post-dose
(Example 9, FIG. 4). When carfilzomib was delivered as liposomal
compositions comprising a peptide epoxyketone compound entrapped in
the liposomes' aqueous core, systemic exposure was extended with
detectable total drug (both encapsulated and released) for up to 24
hours post-dose (Example 9, FIG. 4). These data demonstrate that
the liposomal compositions comprising a peptide epoxyketone
compound entrapped in the liposomes' aqueous core resulted in
significantly greater exposure and longer circulation relative to
an injectable, non-liposomal, peptide epoxyketone compound
composition.
[0327] Also, liposomal compositions of the present invention
maintain anti-tumor efficacy as compared to a non-liposomal
composition comprising the same peptide epoxyketone compound;
further, liposomal compositions of the present invention maintain
anti-tumor efficacy at a reduced dosing frequency as compared to a
non-liposomal composition comprising the same peptide epoxyketone
compound (Example 11, FIG. 7, FIG. 8; Example 12, FIG. 9, FIG.
10).
[0328] Example 13 sets forth exemplary criteria for screening and
selection of advantageous liposomal compositions comprising peptide
epoxyketone compounds. Liposomal compositions of the present
invention and those produced by the methods of the present
invention are screened based on, for example, pharmacokinetic data
(e.g., plasma half-life and area under the plasma concentration
time curve), pharmacodynamic profiles (e.g., biodistribution,
maximum CT-L activity inhibition, and prolonged inhibition in
tissues), and anti-tumor activity (e.g., as evaluated in human
tumor xenograft rodent studies).
[0329] Accordingly, the data in the Examples demonstrate that
liposomal compositions of the present invention resulted in
prolonged exposure without affecting biodistribution. Tolerability
of the peptide epoxyketone compound was also enhanced in animals,
likely due to reduced exposure to high concentrations of free drug.
The liposomal compositions comprising peptide epoxyketone compounds
of the present invention provide the following improvements
relative to the current non-liposomal CFZ SBE-B-CD composition:
improved pharmacodynamic profiles of peptide epoxyketone compounds
by delaying proteasome recovery; improved pharmacokinetic profiles
by decreasing clearance and extending plasma half-life; improved
safety profiles of peptide epoxyketone compounds (i.e.
tolerability); and reduced dosing frequency.
[0330] Experimental
[0331] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to practice the present invention, and are not
intended to limit the scope of what the inventors regard as the
invention. Efforts have been made to ensure accuracy with respect
to numbers used (e.g., amounts, concentrations, percent changes,
etc.) but some experimental errors and deviations should be
accounted for. Unless indicated otherwise, temperature is in
degrees Centigrade and pressure is at or near atmospheric.
[0332] The compositions used to practice the methods of the present
invention meet the specifications for content and purity required
of pharmaceutical products.
1.0 MATERIALS AND METHODS
Particle Size Reduction of Liposomal Suspension
[0333] Particle size reduction or deagglomeration of the rehydrated
liposomal suspension can be carried out either by sonication (20 to
60 minutes) or by high-pressure homogenization/microfluidizer (up
to 30,000 psi).
[0334] Content Determination by HPLC Assay
[0335] The liposomal suspension can be mixed with an organic
solvent, for example, methanol, to dissolve lipids and free the
carfilzomib. The solution can be filtered through a 0.2 .mu.m PTFE
filter prior to HPLC analysis.
[0336] Carfilzomib content can be determined by a gradient HPLC
assay according to the method in Table 1 using sodium perchlorate
buffer, 0.1M, pH 3.1 and acetonitrile (50:50 v/v).
TABLE-US-00003 TABLE 1 Column: Phenomenex Gemini .TM. C18, 150
.times. 4.6 mm, 5 .mu.m particle size Column Temperature: 30 .+-.
2.degree. C. Autosampler Temperature: 5 .+-. 3.degree. C. Detection
Wavelength: 220 nm Flow Rate: 1.5 mL/min Injection Volume: 10 .mu.L
Total Run Time: 17 min
[0337] Liposome Morphology
[0338] The vesicle size, shape and surface morphology of the
liposomal composition can be determined by scanning electron
microscopy (SEM) and transmission electron microscopy (TEM).
[0339] Liposome/Carfilzomib Solid State
[0340] Polarized light microscopy, differential scanning
calorimetry (DSC), X-ray diffraction (XRD), and freeze fracture
electron microscopy can be used to elucidate the phase behavior of
the vesicles.
[0341] Liposome Size and Distribution
[0342] Polarized light microscopy, dynamic light scattering, and
TEM can be used to determine the size and size distribution range
of the liposomes of the liposomal composition.
[0343] Determination of Free Drug
[0344] Because carfilzomib has extremely low aqueous solubility,
the amount of entrapped drug versus free drug can be qualitatively
determined based on polarized light microscope. Free drug
precipitates in the aqueous medium, due to its extremely low
aqueous solubility, and the precipitated material can be seen using
a polarized light microscope.
Sulfobutyl ether-.beta.-cyclodextrins
[0345] Sulfobutyl ether-.beta.-cyclodextrins (SBE-B-CD), for
example, CAPTISOL.RTM., can be synthetically produced and/or are
commercially available, for example from Ligand Pharmaceuticals,
Inc., La Jolla, Calif.
2.0 EXAMPLES
Example 1
Preparation of Molecularly Dispersed Carfilzomib in Thin Lipid
Film
[0346] To make liposomal carfilzomib, the following materials in
the indicated ratios were added to a suitably sized round bottom
flask: drug to total lipid weight ratio of 0.05:0.95 to 0.17:0.83.
Total lipids typically comprise the lipids EPC, HSPC, DSPC, DSPG,
POPC, DPPC, DSPE, and/or sphingomyelin (SPH) alone, or with
cholesterol. If cholesterol is added, the phospholipid to
cholesterol weight ratio (phospholipid:cholesterol) of from 0.9:0.1
to 0.5:0.5. An appropriate volume of phosphate buffer saline was
used to rehydrate the lipid film to give a target carfilzomib
concentration of 1 and 2 mg/mL, respectively.
[0347] To make PEGylated liposomal carfilzomib, the following
materials in the indicated ratios were added to a suitably sized
round bottom flask: drug to total lipid weight ratio of 0.11:0.89
to 0.16:0.84. Phospholipids typically comprise the lipids EPC,
HSPC, DSPC, DPPC, DSPG, POPC, SPH, DSPE, or combinations thereof.
When PEG-derivatized lipids are present (e.g., PEG-derivatized
phospholipids) the typical weight ratio for total lipids
(phospholipids:PEG-derivatized lipids) is from 0.9:0.1 to
0.75:0.25. Further, when cholesterol is also added the typical
weight ratio for total lipids is (phospholipids:PEG-derivatized
lipids:cholesterols) is from 0.833:0.0833:0.0833 to 0.57:0.14:0.29.
An appropriate volume of phosphate buffer saline was used to
rehydrate the lipid film to give a target carfilzomib concentration
of 2 mg/mL.
[0348] To dissolve the lipids and carfilzomib, an appropriate
volume of organic solvent (e.g., cloroform:MeOH (60:40 or 50:50
v/v)), enough to achieve between 10-20 mg/mL dissolved lipid, was
added to the flask. The flask was attached to a rotary evaporator
spinning at 50-100 rpm and immersed in a water bath set above the
highest gel-liquid crystal phase transition (Tc) temperature of the
lipids used. Although the Tc for egg phosphatidylcholine is below
room temperature (-15.degree. C. to -7.degree. C.), the temperature
bath used for EPC was approximately 45.degree. C. to 50.degree. C.
For DSPC, DPPC, and mPEG-DSPE, the water bath temperature should be
set greater than 55.degree. C., 41.degree. C., and 50.degree. C.,
respectively.
[0349] The flask was allowed to rotate in the water bath for
approximately 1 minute to equilibrate. A slow vacuum was pulled, to
as low as <10 Torr, to obtain a thin dry film on the walls of
the flask without precipitation. To remove any residual solvent,
the flask was subjected to high vacuum at room temperature for a
few hours or overnight. Table 2A presents nominal concentrations of
the components of exemplary liposomal carfilzomib compositions
(liposomal carfilzomib compositions, L-CFZ; pegylated liposomal
carfilzomib compositions, pL-CFZ), as well as control compositions
(i.e., "empty" liposomes).
TABLE-US-00004 TABLE 2A mPEG- Composition CFZ* Phospholipid DSPE
Cholesterol Designation Composition Name (mg/mL) (mg/mL) (mg/mL)
(mg/mL) A Empty EPC 0 12.5 0 0 Liposomes B 1 mg/mL Liposomal 1 12.5
0 0 CFZ C 2 mg/mL Liposomal 2 12.5 0 0 (EPC) CFZ D Empty PEG- 0
12.5 1.3 0 Liposomes (EPC) E 2 mg/mL PEG- 2 12.5 1.3 0 Liposomal
(EPC) CFZ F Empty PEG- 0 12.5 1.3 3.1 Liposomes (EPC) w/
Cholesterol G 2 mg/mL PEG- 2 12.5 1.3 3.1 Liposomal (EPC) CFZ w/
Cholesterol H 2 mg/mL PEG- 2 12.5 1.3 3.1 Liposomal (SPH) CFZ w/
Cholesterol *Total drug content may include unencapsulated drug
that was not removed during processing.
[0350] The nominal concentrations of exemplary liposomal
Compositions A to H are presented in Table 2A.
[0351] Note that the drug to lipid ratios in FIG. 1 (Drug:Lipid
Ratio (%)) were calculated by taking the weight of drug
(carfilzomib) divided by the weight of drug plus weight of
phospholipid (wt. drug/(wt. drug+wt. phospholipid)). However, the
more conventional calculation is a ratio of the weight of drug to
the weight of total lipids (e.g. phospholipid, hydrophilic
polymer-derivatized lipid, cholesterol; see, e.g., Table 3). The
more conventional calculation of Drug:Total Lipid Ratio for the
specific experimental formulations in FIG. 1 are presented in Table
2B.
TABLE-US-00005 TABLE 2B Total Drug:Total Composition Composition
CFZ lipids lipid (wt. Lipid Lot Desig. Name (mg) (mg) ratio)
composition.sup.1 6005- A Empty 0 104.1 0.000:1 1:0:0 27B liposomes
6005- B 1 mg/mL 5.8 111.3 0.052:0.948 1:0:0 29C liposomal CFZ 6005-
D Empty PEG- 0 101.7 0.000:1 0.90:0.10:0 35B liposomes 6005- C 2
mg/mL 17.2 112.3 0.153:0.847 1:0:0 45A Liposomal CFZ 6005- C 2
mg/mL 16.5 105.5 0.156:0.844 1:0:0 45B Liposomal CFZ 6005- C 2
mg/mL 16.85 108.9 0.155:0.845 1:0:0 45A/B Liposomal CFZ Pooled
6005- E 2 mg/mL PEG- 17 107.3 0.158:0.842 0.88:0.12:0 45C liposomal
CFZ 6005- E 2 mg/mL PEG- 16 105.4 0.152:0.848 0.89:0.11:0 45D
liposomal CFZ 6005- E 2 mg/mL PEG- 16.5 106.35 0.155:0.845
0.89:0.11:0 45C/D liposomal CFZ Pooled 6005- C 2 mg/mL 22.6 130.6
0.173:0.827 1:0:0 53A Liposomal CFZ 6005- C 2 mg/mL 41.9 251.8
0.166:0.834 1:0:0 53D Liposomal CFZ 6005- C 2 mg/mL 32.25 191.2
0.169:0.831 1:0:0 53A/D Liposomal CFZ Pooled 6005- A Empty 0 129.8
0.000:1 1:0:0 53C Liposomes 6005- C 2 mg/mL 41.7 252.9 0.165:0.835
1:0:0 67B Liposomal CFZ 6005- C 2 mg/mL 21.3 126.9 0.168:0.832
1:0:0 69A Liposomal CFZ 6005- A Empty 0 125.9 0.000:1 1:0:0 69B
Liposomes 6005- F Empty PEG 0 169.1 0.000:1 0.74:0.08:0.18 71D
Liposomes w/ Cholesterol 6005- G 2 mg/mL PEG 20 172.4 0.116:0.884
0.74:0.08:0.18 71E liposomal CFZ w/ cholesterol .sup.1=
(phospholipid wt./total lipid wt.:mPEG-DSPE wt./total lipid
wt.:cholesterol wt./total lipid wt.)
[0352] Table 3 presents example ranges of components to be used in
liposomal carfilzomib compositions of the present invention, as
well as control compositions (i.e., "empty" liposomes). In the
table, preferred phospholipids include EPC, HSPC, DSPC, DPPC, DSPG,
POPC, SPH, DSPE, or combinations thereof
TABLE-US-00006 TABLE 3 Total Lipid Weight Ratio Drug:Total Lipid
(Phospholipid:Hydrophilic Polymer- Weight Ratio derivatized
Lipid:Cholesterol) Composition Type (wt. %) (wt. %) Empty Liposomes
= 0:1 1:0:0 Empty L (0 wt. % drug/100 wt. % (100 wt. %
Phospholipid) Total Lipid) Liposomal CFZ = L- 0.005:0.995 to
0.35:0.65 1:0:0 CFZ (0.5 wt. % drug/95.5 wt. % (100 wt. %
Phospholipid) Total Lipid to 35 wt. % drug/65 wt. % Total Lipid)
Empty Liposomes 0:1 0.9:0:0.1 to 0.5:0:0.5 w/ Cholesterol = (0 wt.
% drug/100 wt. % (90 wt. % Phospholipid/10 wt. % Cholesterol Empty
L Chol Total Lipid) to 50 wt. % Phospholipid/50 wt. % Cholesterol)
Liposomal CFZ w/ 0.005:0.995 to 0.35:0.65 0.9:0:0.1 to 0.5:0:0.5
Cholesterol = L- (0.5 wt. % drug/95.5 wt. % (90 wt. %
Phospholipid/10 wt. % Cholesterol CFZ Chol Total Lipid to 35 wt. %
to 50 wt. % Phospholipid/50 wt. % drug/65 wt. % Cholesterol) Total
Lipid) Empty PEGylated 0:1 0.9:0.1:0 to 0.75:0.25:0 Liposomes =
Empty (0 wt. % drug/100 wt. % (90 wt. % Phospholipid/10 wt. %
Hydrophilic pL Total Lipid) polymer-derivatized lipid to 75 wt. %
Phospholipid/25 wt. % Hydrophilic polymer- derivatized lipid)
PEGylated 0.005:0.995 to 0.35:0.65 0.9:0.1:0 to 0.75:0.25:0
Liposomal CFZ = (0.5 wt. % drug/95.5 wt. % (90 wt. %
Phospholipid/10 wt. % Hydrophilic pL-CFZ Total Lipid to 35 wt. %
polymer-derivatized lipid to 75 wt. % drug/65 wt. % Phospholipid/25
wt. % Hydrophilic polymer- Total Lipid) derivatized lipid) Empty
PEGylated 0:1 0.833:0.0833:0.0833 to 0.57:0.14:0.29 Liposomes w/ (0
wt. % drug/100 wt. % (83.3 wt. % Phospholipid/8.33 wt. %
Cholesterol = Empty Total Lipid) Hydrophilic polymer-derivatized
lipid/8.33 wt. % pL Chol Cholesterol to 57 wt. % Phospholipid/ 14
wt. % hydrophilic polymer-derivatized lipid/29 wt. % Cholesterol
PEGylated 0.005:0.995 to 0.35:0.65 0.833:0.0833:0.0833 to
0.57:0.14:0.29 Liposomal CFZ w (0.5 wt. % drug/95.5 wt. % (83.3 wt.
% Phospholipid/8.33 wt. % Cholesterol = pL- Total Lipid to 35 wt. %
Hydrophilic polymer-derivatized lipid/8.33 wt. % CFZ Chol drug/65
wt. % Cholesterol to 57 wt. % Phospholipid/ Total Lipid) 14 wt. %
Hydrophilic polymer-derivatized lipid/29 wt. % Cholesterol
Example 2
Lipid Hydration
[0353] The thin-filmed, round-bottom flask was immersed in a water
bath set above the highest gel-liquid crystal phase transition.
When EPC was used, rehydration occurred at room temperature. For
DSPC, DPPC, and mPEG-DSPE, the water bath temperature should be set
greater than 55.degree. C., 41.degree. C., and 50.degree. C.,
respectively. An appropriate volume of phosphate buffered saline,
pH 7.2, or water for injection was added to the lipid film to
achieve the desired target carfilzomib concentration or dose. The
flask was mildly agitated or shaken with intermittent vortexing, as
needed, and sonicated in water bath at the appropriate Tc
temperature for 1 to 2 minutes to facilitate complete hydration
from flask walls.
[0354] After the film was dispersed, the mixture was transferred to
a vial and sonicated for an additional 20 to 40 minutes in a water
bath above the Tc to size the liposomes. When EPC was used, the
temperature of the water bath in the sonicator was kept near room
temperature. Upon hydration the lipid suspension appeared as a
slightly hazy or milky solution.
[0355] FIG. 1 sets forth the composition of exemplary liposomal
carfilzomib compositions of the present invention, as well as
control compositions (i.e., "empty" liposomes) used in the studies
described below. The nominal concentrations of exemplary liposomal
Compositions A to G are presented in Table 2A.
Example 3
Characterization of Liposomes
[0356] Particle size reduction and/or deagglomeration of the
rehydrated liposomal suspension was carried out by sonication (20
to 60 minutes).
[0357] The CFZ content of exemplary liposomal carfilzomib
compositions was determined by HPLC as described above. The
liposomal compositions were each diluted in methanol to dissolve
lipids and carfilzomib. The solution was filtered through a 0.2
.mu.m PTFE filter prior to HPLC analysis. The percent difference
between the theoretical and experimental liposomal drug
concentrations for the prepared lots (FIG. 1) were typically 2% or
less (except for one of the first lots which had a 12% difference).
The results of the HPLC analysis are presented in FIG. 1.
[0358] Based on polarized light microscope it was qualitatively
determined that nearly all of the drug was entrapped in the
liposomes. Any free drug would precipitate in the aqueous medium
due to its extremely low aqueous solubility of <1 .mu.g/mL and
any precipitated material could be easily observed under the
polarized light microscope. No visible precipitate was observed
under the polarized light microscope.
Example 4
Tolerability of Liposomal Carfilzomib
[0359] The tolerability of carfilzomib incorporated in either
liposomal (L-CFZ) or pegylated liposomal (pL-CFZ) carfilzomib
compositions was evaluated in both mice and rats above the maximum
tolerated dose (MTD) achieved using an injectable composition of
carfilzomib formulated in 10% sulfobutylether beta cyclodextrin
(SBE-.beta.-CD, also referred to as CFZ SBE-B-CD), 10 mM Citrate,
pH 3.5 (see, e.g., U.S. Patent Publication Nos. 2011/0236428).
[0360] Liposomal compositions prepared in Example 2 and
characterized in Example 3 were rehydrated with an appropriate
volume of aqueous medium to achieve a target carfilzomib
concentration in the range of approximately 1 to 2 mg/ml (see
composition data in FIG. 1). Toxicity for both L-CFZ and pL-CFZ
compositions were tested in mice (Table 4). Toxicity for L-CFZ was
tested in rats (Table 5).
[0361] Female BALB/c mice (7-8 week old; 5/cohort) were dosed
intravenously as follows: 15 mg/kg CFZ SBE-B-CD (7.5 mL/kg); 10
mg/kg L-CFZ (5 mL/kg); 10 mg/kg pL-CFZ (5 mL/kg); 15 mg/kg L-CFZ
(7.5 mL/kg); 15 mg/kg pL-CFZ (7.5 mL/kg); 20 mg/kg L-CFZ (10
mL/kg); 20 mg/kg pL-CFZ (10 mL/kg); 25 mg/kg L-CFZ (12.5 mL/kg); 25
mg/kg pL-CFZ (12.5 mL/kg); 30 mg/kg L-CFZ (15 mL/kg); 35 mg/kg
L-CFZ (17.5 mL/kg); or empty liposome (15 mL/kg).). Survival was
then monitored over a seven day period. The survival rates of mice,
the liposomal compositions, and dosing used for treatment were as
shown in Table 4.
TABLE-US-00007 TABLE 4 Overall Composition Desig. (Dose Dose
Mortality Group Volume) (mg/kg) (No. dead/total) CFZ alone 2 mg/mL
SBE-B-CD 15 5/5 (10 mg/kg MTD) Composition (150 .mu.L) Empty
Liposome Composition A (300 .mu.L) 0 0/5 L-CFZ Composition B (200
.mu.L) 10 0/5 L-CFZ Composition C (150 uL) 15 0/5 L-CFZ Composition
C (200 uL) 20 0/5 L-CFZ Composition C (250 uL) 25 0/5 L-CFZ
Composition C (300 uL) 30 1/5 L-CFZ Composition C (350 uL) 35 4/5
Empty PEG Composition D (300 .mu.L) 0 0/5 Liposome pL-CFZ
Composition E (100 uL) 10 0/5 pL-CFZ Composition E (150 uL) 15 0/5
pL-CFZ Composition E (200 uL) 20 5/5 pL-CFZ Composition E (250 uL)
25 5/5
[0362] Male Sprague Dawley rats (5/cohort) weighing approximately
250-300 grams were dosed intravenously with the following: 8 mg/kg
CFZ (SBE-B-CD composition) (5 mL/kg); 8 mg/kg L-CFZ (4 mL/kg); 10
mg/kg L-CFZ (5 mL/kg); 12.5 mg/kg L-CFZ (6 mL/kg); or empty
liposome (5 mL/kg). Survival was then monitored over a seven day
period. The survival rates of dosed rats were as shown in Table
5.
TABLE-US-00008 TABLE 5 Overall Mortality Dose (No. Group
Composition Desig. (mL/kg) (mg/kg) dead/total) CFZ SBE-B-CD
Composition 8 2/5 (7 mg/kg MTD) (5 mL/kg) Empty Liposome
Composition A (5 mL/kg) 0 0/5 L-CFZ Composition C (4 mL/kg) 8 0/5
L-CFZ Composition C (5 mL/kg) 10 0/5 L-CFZ Composition C (6.25
mL/kg) 12.5 3/5
[0363] Liposomal carfilzomib significantly enhanced tolerability
(Table 4) by increasing the maximum tolerated dose (MTD) of
carfilzomib in mice by approximately 2.5 fold for the liposomal
carfilzomib compositions and by 0.5 fold with PEGylated liposomal
carfilzomib compositions compared to SBE-B-CD based carfilzomib
composition. Only a slight increase in the MTD in rats was observed
with liposomal carfilzomib L-CFZ (10 mg/kg) compared to carfilzomib
(7 mg/kg) formulated in SBE-B-CD.
[0364] These data demonstrate that liposomal compositions
comprising peptide epoxyketone compounds significantly enhanced
tolerability by increasing the maximum tolerated dose (MTD) of a
peptide epoxyketone compound relative to non-liposomal compositions
comprising peptide epoxyketone compounds.
Example 5
Pharmacodynamic Response of CFZ Liposomal Compositions
[0365] The pharmacodynamic response of carfilzomib formulated in
SBE-B-CD (CFZ SBE-B-CD) (using a non-liposomal, injectable
composition of carfilzomib formulated in 10% sulfobutylether beta
cyclodextrin (SBE-B-CD), 10 mM Citrate, pH 3.5 (see, e.g., U.S.
Patent Publication Nos. 2011/0236428)), empty liposomes
(Composition D), liposomes comprising CFZ (L-CFZ, Composition C),
and pegylated liposomes comprising (pL-CFZ, Composition E) was
evaluated in BALB/C mice following a single intravenous bolus
administration.
[0366] The mice (three mice per time point) were administered a
dose of 10 mg/kg in a dose volume of 5 mL/kg. Blood samples and
tissues for pharmacodynamic testing were taken at 1, 8, and 24
hours after administration of each composition. The pharmacodynamic
response was determined by measurement of proteasome activity in
whole blood (primarily erythrocytes) (see FIG. 2A), adrenal (see
FIG. 2B), liver (see FIG. 2C), and heart (see FIG. 2D), using a
fluorogenic substrate (LLVY-AMC [Leu-Leu-Val-Tyr-AMC {AMC=7-amido
4-methylcoumarin}]; as described by Lightcap E S, McCormack T A,
Pien C S, et al., Clin. Chem. 46:673-683 (2000)) to quantitate the
chymotrypsin-like activity of the proteasome. All samples were
normalized to the appropriate vehicle (i.e., the corresponding
composition without CFZ), and the vehicle time point was 1 hour
post dose. Three tissue samples were evaluated per time point for
each tissue from each mouse.
[0367] A single intravenous dose of 10 mg/kg resulted in rapid
proteasome inhibition of >80% within 1 hour in whole blood and
all tissues. Similar and complete recovery from proteasome
inhibition was observed 24 hours post-dose in all tissues tested
except for the blood and heart and occurred with at t.sub.1/2 of
8-24 hours for all compositions. The slower recovery observed in
the heart with both the liposomes and pegylated liposomes suggest
that the heart tissue may act as a depot. As expected, there was no
recovery of proteasome activity in blood due to the irreversible
binding of carfilzomib and the lack of the erythrocytes to
synthesize new proteasome.
[0368] These observations indicate that inhibition of proteasome
activity in whole blood and tissues is rapid, similar across
compositions. The liposomal compositions did not adversely affect
biodistribution of CFZ.
Example 6
Circulation Half-Life of Liposomal CFZ
[0369] Circulation half-life of liposomal CFZ was evaluated in 7-8
week old female BALB/c mice (3/time point) following a single i.v.
injection of either 5 mg/kg CFZ formulated in 10% sulfobutylether
beta cyclodextrin, 10 mM Citrate, pH 3.5 (non-liposomal) or 15
mg/kg of liposomal carfilzomib compositions.
[0370] When CFZ was delivered in the composition containing
SBE-B-CD at 5 mg/kg, plasma concentration rapidly declines with
time and drops to below the limit of quantitation (BLOQ; limit of
quantitation--LOQ) after 60 minutes (Table 6). The terminal plasma
half-life (t.sub.1/2) was about 20 minutes.
TABLE-US-00009 TABLE 6 Plasma Levels of CFZ Using SBE-B-CD
Composition I.V. Bolus 5 mg/kg in BALB/c Mice Plasma Conc. (uM)
Time (min) Mean STD 0 0 0 2 10.379 0.844 5 1.732 0.431 10 0.310
0.064 20 0.176 0.040 30 0.061 0.011 60 0.042 0.032 LOQ = 1 ng/mL
(MW = 719.4)
[0371] When delivered in liposomal compositions at 15 mg/kg (using
L-CFZ, Composition C, or pL-CFZ-Chol, Composition G, with a dose
volume of 150 .mu.L), detectable CFZ was observed at 6 hours
post-dosing (Table 7).
TABLE-US-00010 TABLE 7 Mean Plasma Levels of Liposomal CFZ IV bolus
at 15 mg/kg in BALB/c Mice Plasma Conc. (uM) Plasma Conc. (uM)
Liposomal PEGylated CFZ Liposomal CFZ Composition C Composition G
Time (min) Mean STD Mean STD predose BLOQ BLOQ BLOQ BLOQ 2 102 21
79.0 22.7 5 51.4 9.1 42.5 8.4 10 22.2 6.2 9.81 3.48 30 1.26 0.48
0.183 0.045 60 0.143 0.018 0.0537 0.0200 120 0.0424 0.0134 0.0111
0.0010 240 0.0125 0.0010 0.0152 0.0070 360 0.0129 0.0023 0.0122
0.0100
[0372] The t.sub.1/2 was 140 and 201 minutes, respectively, for
liposomal CFZ and pegylated liposomal CFZ compositions,
respectively. The data for liposomal CFZ compositions versus CFZ
SBE-B-CD, clearly demonstrate the ability of liposome to
significantly enhance the circulation half-life of peptide
epoxyketone compounds.
[0373] Circulation half-life of liposomal CFZ was also evaluated in
male Sprag-Dawley rats (3/time point) weighing approximately
250-300 grams following a single i.v. injection of 8 mg/kg CFZ
formulated in 10% sulfobutylether beta cyclodextrin, 10 mM Citrate,
pH 3.5, or 8 mg/kg liposomal CFZ. Similar to mice, a rapid decline
in plasma concentration was observed in rats when CFZ SBE-B-CD was
delivered (Table 8), with a plasma t.sub.1/2 of 17 minutes. When
CFZ was delivered in liposomal compositions (L-CFZ, Composition C,
FIG. 1, with a dose volume of 4 mL/kg) at the same dose level,
detectable CFZ was observed at 4 hours post-dosing (Table 8), with
a t.sub.1/2 of about 50 minutes.
TABLE-US-00011 TABLE 8 Plasma Levels of CFZ Using SBE-B-CD
Composition or Liposomal CFZ (IV bolus at 8 mg/kg) in Rats SBE-B-CD
Liposomal-CFZ Composition Composition C Plasma Conc. (uM) Time
(min) Mean STD Mean STD predose BLOQ BLOQ BLOQ BLOQ 0.1 42.9 4.4 ND
ND 1 3.93 0.36 ND ND 2 1.90 0.26 36.9 4.8 5 0.651 0.115 21.0 1.8 15
0.0505 0.0030 0.583 0.072 30 0.0189 0.0030 0.139 0.050 60 0.0072
0.0020 0.058 0.029 120 ND ND 0.012 0.003 240 ND ND 0.004 0.001 420
ND ND BLOQ BLOQ
[0374] The data presented in Table 9 and Table 10 demonstrate that,
compared to CFZ SBE-B-CD, the exposure (AUC) to liposomal CFZ
compositions (L-CFZ Composition C) and pegylated liposomal CFZ
compositions (pL-CFZ Composition E), was increased about 5 to 7 and
20-fold in mice and rats, respectively.
TABLE-US-00012 TABLE 9 Mean AUC Levels of CFZ Using SBE-B-CD
Composition or Liposomal CFZ (IV bolus at 15 mg/kg) in Mice
Relative Increase Dose AUCinf AUC-.sub.liposome/ Species (mg/kg)
Composition (min * .mu.mol/L) AUC-.sub.SBE-B-CD Mouse 15 SBE-B-CD
130.5 -- Mouse 15 L-CFZ-C 942.8 7 Mouse 15 pL-CFZ-E 623.3 5
TABLE-US-00013 TABLE 10 Mean AUC Levels of CFZ Using SBE-B-CD
Composition or Liposomal CFZ (IV bolus at 8 mg/kg) in Rats Relative
Increase Dose AUCinf AUC-.sub.liposome/ Species (mg/kg) Composition
(min * .mu.mol/L) AUC-.sub.SBE-B-CD Rat 8 SBE-B-CD 14.5 -- Rat 8
L-CFZ-C 297.7 20
[0375] The increased AUC is significant because AUC is determined
by measuring drug clearance rates and these data demonstrate that
the liposomal compositions of the present invention are decreasing
clearance of peptide epoxyketone compounds. These data demonstrate
the extended duration of exposure to peptide epoxyketone compounds
in liposomal composition versus the non-liposomal SBE-B-CD
composition.
Example 7
Preparation of Thin Lipid Film and Use in Preparing Liposomes
Comprising an Aqueous Core Loaded with Peptide Epoxyketone
Compounds Complexed with SBE-B-CD
[0376] To make the PEGylated liposomal film, the following
materials at their indicated ratios were added to a suitably sized
round bottom flask. Total lipids typically comprise the lipids EPC,
HSPC, DSPC, DPPC, DSPG, POPC, SPH, DSPE, or combinations thereof,
with PEG-derivatized lipids (e.g., PEG-derivatized phospholipids)
in weight ratio (phospholipids:PEG-derivatized lipids) of from
0.9:0.1 to 0.75:0.25, or when cholesterol is added the phospholipid
to PEG-derivatized lipid to cholesterol weight ratio
(phospholipid:PEG-derivatized lipid:cholesterol) is from
0.83:0.083:0.083 to 0.57:0.14:0.29.
[0377] To dissolve the lipids, an appropriate volume of organic
solvent (e.g., cloroform:MeOH (60:40 v/v)), enough to achieve
between 10-20 mg/mL dissolved lipid, was added to the flask. The
flask was attached to a rotary evaporator spinning at 100 rpm and
immersed in a water bath set above the highest gel-liquid crystal
phase transition (Tc) temperature of the lipids used. Although the
Tc for egg phosphatidylcholine is below room temperature
(-15.degree. C. to -7.degree. C.), the temperature bath used for
EPC was approximately 45.degree. C. to 50.degree. C. For DSPC,
DPPC, and mPEG-DSPE, the water bath temperature should be set
greater than 55.degree. C., 41.degree. C., and 50.degree. C.,
respectively. If there is no phase transition temperature, the
water bath temperature is set between 35-45.degree. C.
[0378] The flask was allowed to rotate in the water bath for
approximately 1 minute to equilibrate. A slow vacuum was pulled, to
as low as <10 Torr, to obtain a thin dry film on the walls of
the flask without precipitation (typically for about 30 minutes).
To remove any residual solvent, the flask was subjected to high
vacuum at room temperature for a few hours or overnight.
[0379] Carfilzomib (CFZ) was solubilized in an aqueous solution by
complexation with sulfobutylether beta cyclodextrin (SBE-B-CD).
Excess carfilzomib was added to an aqueous solution of 20% SBE-B-CD
and 20 mM citric acid. The solution pH was adjusted to
approximately pH 2.5 with 1N HCl, if needed to solubilize CFZ. The
mixture was sonicated for approximately 10 minutes and stirred
using a magnetic stir bar for not less than an hour prior to
filtration through a 0.2 .mu.m filter to remove excess undissolved
drug. After filtration the solution pH was adjusted to between pH
3.5 and 5. This aqueous solution of CFZ complexed with SBE-B-CD was
used to rehydrate the thin lipid film.
[0380] Once the vesicles were rehydrated the unencapsulated free
drug was removed by centrifugation at 31000 rpm for 30 minutes and
washing with PBS or by being dialyzed using a membrane with a MWCO
of 8-10 kD in PBS for up to 48 hours.
[0381] Table 11 presents nominal concentrations of the components
of exemplary liposomal compositions comprising liposomes comprising
an aqueous core loaded with CFZ complexed with SBE-B-CD.
TABLE-US-00014 TABLE 11 Total Lipid Composition Weight Drug:Total
Designation Lipid composition Ratios Lipid Ratio* Drug content**
apL 12.5 mg/mL EPC, 0.73:0.08:0.19 0.05:0.95 0.9 mg/mL 1.3 mg/mL
mPEG- DSPE, 3.3 mg/mL cholesterol apL-9 25 mg/mL EPC,
0.73:0.08:0.19 0.04:0.96 1.3 mg/mL 2.5 mg/mL mPEG- DSPE, 6.3 mg/mL
cholesterol apL-11 (for 12.5 mg/mL egg 0.73:0.08:0.19 0.05:0.95 0.8
mg/mL 15 mg/kg SPH, 1.3 mg/mL dosing) mPEG-DSPE, 3.3 mg/mL
cholesterol apL-11 (for 12.5 mg/mL egg 0.73:0.08:0.19 0.01:0.99 0.2
mg/mL 5 mg/kg SPH, 1.3 mg/mL dosing) mPEG-DSPE, 3.3 mg/mL
cholesterol *assuming 100% drug encapsulation **Total drug content
may include unencapsulated drug that was not removed during
processing.
Example 8
Pharmacodynamic Response of CFZ Liposomal Compositions Comprising
Liposome Comprising an Aqueous Core Loaded with Peptide Epoxyketone
Compounds Complexed with SBE-B-CD
[0382] The pharmacodynamic response of liposomal compositions
comprising liposomes comprising an aqueous core loaded with CFZ
complexed with SBE-B-CD was evaluated in BALB/C mice following a
single intravenous bolus administration.
[0383] The pharmacodynamic response of injectable carfilzomib
formulated in SBE-B-CD (non-liposomal; see, e.g., U.S. Patent
Publication Nos. 2011/0236428) or liposomal compositions comprising
liposomes comprising an aqueous core loaded with CFZ complexed with
SBE-B-CD was evaluated in BALB/C mice (apL-11 (for 15 mg/kg
dosing), Example 7; and apL-11 (for 5 mg/kg dosing), Example 7)
following a single intravenous bolus administration. The mice
(three mice per time point) were administered a dose of 5 or 10
mg/kg of non-liposomal carfilzomib or 5 or 15 mg/kg of liposomal
carfilzomib as a solution in a dose volume of 5 mL/kg. Blood
samples and tissues for pharmacodynamic testing were taken at 0, 1,
8, and 14 hours after administration of the non-liposomal
composition at 10 mg/kg; 0, 1, 4, 6, 8 and 24 hours after
administration of the non-liposomal composition at 5 mg/kg; and 0,
1, 4, 6, and 24 hours after administration of the liposomal
compositions at 5 mg/kg and 15 mg/kg. Three tissue samples were
evaluated per time point for each tissue from each mouse. The
pharmacodynamic response was determined by measurement of
proteasome activity in whole blood (primarily erythrocytes),
adrenal, liver, and heart using a fluorogenic substrate (LLVY-AMC;
as described by Lightcap E S, McCormack T A, Pien C S, et al.,
Clin. Chem. 46:673-683 (2000)) to quantitate the chymotrypsin-like
activity of the proteasome. All samples were normalized to the
corresponding vehicle without CFZ, and the vehicle sample time
point measurement was 1 hour post dose.
[0384] A single dose of injectable carfilzomib formulated in
SBE-B-CD (non-liposomal) at either 5 or 10 mg/kg or liposomal
compositions comprising liposomes comprising an aqueous core loaded
with CFZ complexed with SBE-B-CD (apL-11) at either 5 or 15 mg/kg
resulted in a rapid inhibition of proteasome activity within 1 hour
in whole blood and all other tissues. Greater inhibition of
proteasome activity was observed at the 15 mg/kg dose, which
resulted in >80% inhibition of proteasome activity at 1 hour in
all tissue: whole blood (primarily erythrocytes) (see FIG. 3A),
heart (see FIG. 3B), liver (see FIG. 3C), and adrenal (see FIG.
3D). Similar and near complete recovery from proteasome inhibition
was observed 24 hours post-dose in all tissues tested except for
the blood and heart and occurred with at t.sub.1/2 of 6-24 hours
for both the 5 and 10 mg/kg dose levels of injectable carfilzomib
formulated in SBE-B-CD (non-liposomal). Delayed recovery of
proteasome activity in the adrenals was observed with liposomal
compositions comprising liposomes comprising an aqueous core loaded
with CFZ complexed with SBE-B-CD at both 5 and 15 mg/kg; this
result suggests long term exposure of CFZ. As expected, there was
no recovery of proteasome activity in blood due to the irreversible
binding of carfilzomib and the lack of the erythrocytes to
synthesize new proteasome.
[0385] These observations indicate that inhibition of proteasome
activity in whole blood and tissues was rapid and similar across
between the non-liposomal composition and liposomal compositions
comprising peptide epoxyketone compounds. Further, the delay in the
recovery of proteasome activity in the adrenals with liposomal
compositions comprising liposomes comprising an aqueous core loaded
with peptide epoxyketone compound complexed with SBE-B-CD suggests
extended exposure with the liposomal composition versus the
non-liposomal composition. Further, the liposomal compositions
comprising peptide epoxyketone compounds did not adversely affect
biodistribution of the peptide epoxyketone compounds.
Example 9
Circulation Half-Life of Liposomal CFZ
[0386] Circulation half-life of liposomal CFZ was evaluated in 7-8
week old female BALB/c mice (3/time point) following a single i.v.
injection of the following: 5 mg/kg CFZ formulated in 10%
sulfobutylether beta cyclodextrin (CAPTISOL.RTM.), 10 mM Citrate,
pH 3.5 (CFZ SBE-B-CD; non-liposomal); 5 mg/kg of liposomal
carfilzomib compositions apL-11 (Example 7; apL-11 (for 5 mg/kg
dosing)) and 15 mg/kg of liposomal carfilzomib compositions apL-11
(Example 7; apL-11 (for 15 mg/kg dosing)).
[0387] As shown in FIG. 4, the plasma concentration of injectable
carfilzomib SBE-B-CD composition (non-liposomal) declined rapidly
following intravenous, bolus administration due to rapid and
extensive metabolism (FIG. 4: open squares containing an X
corresponds to administration of 5 mg/kg of an injectable
carfilzomib SBE-B-CD composition (non-liposomal). The half-life of
carfilzomib dosed at 5 mg/kg was about 20 minutes and carfilzomib
was not detectable after 1 hour post-dose.
[0388] When delivered in liposomal compositions, the duration of
exposure to carfilzomib was greatly extended (FIG. 4, solid squares
correspond to administration of 5 mg/kg of apL11 (Example 7), a
pegylated liposomal composition of carfilzomib wherein the aqueous
core of the pegylated liposomes comprises carfilzomib and SBE-B-CD;
solid circles correspond to administration of 15 mg/kg of apL11
(Example 7), a pegylated liposomal composition of carfilzomib
wherein the aqueous core of the pegylated liposomes comprises
carfilzomib and SBE-B-CD). Total drug (encapsulated and released)
was detectable for up to 24 hours post-dose. This is consistent
with the observed delay in proteasome recovery in tissues.
[0389] The data for liposomal CFZ compositions versus CFZ SBE-B-CD
(non-liposomal), demonstrate the ability of liposomal compositions
to significantly enhance the circulation half-life of peptide
epoxyketone compounds. Further, the data show the ability to
provide extended duration of exposure to peptide epoxyketone
compounds in liposomal compositions versus the non-liposomal
SBE-B-CD composition.
Example 10
Preparation of Liposomes Comprising an Aqueous Core Loaded with
Peptide Epoxyketone Using pH Control and an Ethanol Injection
Method
[0390] To make the liposomal compositions using an ethanol
injection method, the following materials at their indicated ratios
were used. Phospholipids typically comprise the lipids EPC, HSPC,
DSPC, DPPC, DSPG, POPC, SPH, DSPE, or combinations thereof. When
PEG-derivatized lipids are present (e.g., PEG-derivatized
phospholipids) the typical weight ratio for total lipids
(phospholipids:PEG-derivatized lipids) is from 0.9:0.1 to
0.75:0.25. Further, when cholesterol is also added the typical
weight ratio for total lipids (phospholipids:PEG-derivatized
lipids:cholesterols) is from 0.833:0.0833:0.0833 to
0.57:0.14:0.29.
[0391] Other materials used in the ethanol injection method include
the following: absolute Ethanol; 1N HCl; Carfilzomib (crystalline
or amorphous); Hamilton Syringe Gastight, 22 gauge; Dialysis kit,
Spectra/Por.RTM. Float-A-Lyzer.RTM. G2 (Spectrum Laboratories Inc.,
Rancho Dominguez, Calif.) molecular weight cut off (MWCO) 8-10 kD;
Water for Injection (WFI); and Phosphate buffer saline 1X
(PBS).
[0392] A lipid/ethanol solution was prepared as follows: 2 mL of
ethanol containing 125 mg/mL egg sphingomyelin, 31.25 mg/mL
cholesterol, 12.5 mg/mL mPEG-DSPE. If needed, the lipid/ethanol
solution was sonicated several minutes to facilitate
dissolution.
[0393] An aqueous solution of CFZ was prepared as follows: 10 mL of
a 0.1M HCl aqueous solution was prepared (.about.pH 1) and CFZ in
excess of solubility was added. The aqueous solution was sonicated
in heated water bath (.about.30.degree. C.) for 20-30 minutes.
Approximate carfilzomib solubility at pH 1 is 1.8 mg/mL.
Undissolved excess drug was removed by filtering through a 0.2
.mu.m filter to yield a visibly clear solution.
[0394] Alternatively, a supersaturated solution of carfilzomib was
prepared by dissolving amorphous carfilzomib in 0.1M HCl solution
with 6% (v/v) ethanol as a cosolvent followed by sonication in a
warm water bath until the solution became clear.
[0395] Liposomes were formed by rapid injection of 1 mL of the
lipid-ethanol solution into 9 mL of the aqueous solution of CFZ
(prepared by one of the methods just described) with stirring using
a magnetic stir bar. Stirring was continued for 5-10 minutes. The
solution pH was .about.pH 1. The resulting solution was dialyzed
against phosphate buffer saline (or WFI) using the Dialysis kit
(Spectra/Por.RTM. Float-A-Lyzer.RTM. G2) MWCO 8-10 kD for 12 to 16
hours. The bulk dialysis solution was replaced with fresh PBS or
WFI after approximately 6-8 hours. Solution pH of the dialyzed
liposome containing composition was about pH 3 to 3.5. The pH of
the aqueous solution comprising the liposomes was adjusted with
sodium hydroxide to between pH 3.5 to a physiologic pH, .about.pH
6.8.
[0396] Table 12 presents nominal concentrations of the components
of exemplary liposomal compositions.
TABLE-US-00015 TABLE 12 Composition Total Lipid Drug:Total Drug
Designation Lipid composition Weight Ratios Lipid Ratio* content**
apL-15 25 mg/mL egg SPH, 2.5 mg/mL 0.74:0.07:0.19 0.02:0.98 0.6
mg/mL mPEG-DSPE, 6.3 mg/mL cholesterol apL-11b 12.5 mg/mL EPC, 1.2
mg/mL 0.74:0.08:0.18 0.06:0.94 1 mg/mL mPEG-DSPE, 3.1 mg/mL
cholesterol *assuming 100% drug encapsulation **Total drug content
may include unencapsulated drug that was not removed during
processing.
Example 11
Liposomes Comprising Entrapped Peptide Epoxyketone Induce
Anti-Tumor Response
[0397] To evaluate the anti-cancer effect of liposomal compositions
comprising liposomes comprising peptide epoxyketone compounds, an
exemplary liposomal composition was tested in a mouse xenograft
tumor model.
[0398] The liposomal composition was made by the methods described
in Example 1 and Example 2. The liposomal composition was as
follows: pL6 (a specific formulation of Composition H; Table 2A)=2
mg/mL CFZ, 12.5 mg/mL Sphingomylin, 3.2 mg/mL cholesterol, 1.3
mg/mL mPEG-DSPE.
[0399] First, the pharmacodynamic response of injectable
carfilzomib formulated in SBE-B-CD (non-liposomal; see, e.g., U.S.
Patent Publication Nos. 2011/0236428) and the pL-6 a liposomal
composition comprising liposomes loaded with CFZ was evaluated in
BALB/C mice following a single intravenous bolus
administration.
[0400] The mice (three mice per time point) were administered a
dose of 10 mg/kg carfilzomib formulated in SBE-B-CD (non-liposomal)
or 15 mg/kg of pL-6 a liposomal composition comprising liposomes
loaded with CFZ as a solution in a dose volume of 7.5 mL/kg. Blood
samples and tissues for pharmacodynamic testing were taken at 0, 1,
4, 6 and 24 hours after administration of the liposomal composition
and 0, 1, 8, and 24 hours for the non-liposomal composition. Three
tissue samples were evaluated per time point for each tissue from
each mouse. The pharmacodynamic response was determined by
measurement of proteasome activity in whole blood (primarily
erythrocytes), adrenal, liver, and heart using a fluorogenic
substrate (LLVY-AMC; as described by Lightcap E S, McCormack T A,
Pien C S, et al., Clin. Chem. 46:673-683 (2000)) to quantitate the
chymotrypsin-like activity of the proteasome. All samples were
normalized to the corresponding vehicle without CFZ, and the
vehicle sample time point measurement was 1 hour post dose.
[0401] A single dose of injectable carfilzomib formulated in
SBE-B-CD (non-liposomal) at 10 mg/kg (MTD) or liposomal
compositions comprising liposomes comprising entrapped CFZ (pL-6)
at 15 mg/kg resulted in a rapid inhibition of >80% of proteasome
activity within 1 hour in whole blood and all other tissues: whole
blood (primarily erythrocytes) (see FIG. 5A), heart (see FIG. 5B),
liver (see FIG. 5C), and adrenal (see FIG. 5D). Similar and near
complete recovery from proteasome inhibition was observed 24 hours
post-dose in all tissues tested except for the blood and heart and
occurred with a t.sub.1/2 of 6-24 hours for the non-liposomal
injectable CFZ. Delayed recovery of proteasome activity in the
adrenals was observed with the liposomal composition pL-6
suggesting long-term exposure to CFZ. As expected, there was no
recovery of proteasome activity in blood due to the irreversible
binding of carfilzomib and the lack of the erythrocytes to
synthesize new proteasome.
[0402] These observations indicate that inhibition of proteasome
activity in whole blood and tissues is rapid and similar across
compositions. The delay in recovery of proteasome activity in the
adrenals suggests extended exposure with the liposomal composition.
The liposomal compositions did not adversely affect biodistribution
of CFZ.
[0403] Second, circulation half-life of liposomal CFZ was evaluated
in 7-8 week old female BALB/c mice (3/time point) following a
single i.v. injection of the following: injectable carfilzomib
formulated in SBE-B-CD (non-liposomal) administered at 5 mg/kg; and
the pL-6 a liposomal composition comprising liposomes loaded with
CFZ administered at 15 mg/kg.
[0404] As shown in FIG. 6, the plasma concentration of injectable
carfilzomib SBE-B-CD composition (non-liposomal) declined rapidly
following intravenous, bolus administration and was below the limit
of quantitation after 1 hour. The half life was about 20 minutes.
This is due to rapid and extensive metabolism (FIG. 6, line with
open circles).
[0405] When delivered in the pL-6 liposomal composition, the
duration of exposure to carfilzomib was greatly extended (FIG. 6,
solid squares). Plasma concentration of total drug (encapsulated
and released) declined slowly and was detectable for up to 24 hours
post-dose. This is consistent with the observed delay in proteasome
recovery in tissues.
[0406] The data for the liposomal CFZ compositions versus CFZ
SBE-B-CD (non-liposomal) demonstrate the ability of liposomal
compositions to significantly enhance the circulation half-life of
peptide epoxyketone compounds. Further, the data show the ability
to provide extended duration of exposure to peptide epoxyketone
compounds in liposomal compositions versus the non-liposomal
SBE-B-CD composition.
[0407] Third, the anti-tumor response of injectable carfilzomib
formulated in SBE-B-CD (non-liposomal) and the pL-6 liposomal
composition comprising liposomes loaded with CFZ was evaluated in
mice. Tumors were established by s.c. injection of RL cells (human
non-Hodgkin's B cell lymphoma cells; passage number <9 and
viability >95% at the time of implantation) in the right flank
of beige-nude-XID (BNX) mice (n=8 per group). For RL studies, cell
suspensions containing 1.times.10.sup.7 cells in a volume of 0.1 mL
were injected. Mice were randomized into treatment groups and
dosing initiated when tumors reached .about.100 mm.sup.3 (RL).
Tumors were measured thrice weekly by recording the longest
perpendicular diameters and tumor volumes were calculated using the
equation V (in mm.sup.3)=(length X width)/2.
[0408] BNX mice bearing established human tumor xenograft derived
from RL cells were treated with either non-liposomal carfilzomib or
liposomal carfilzomib. Drug was administered on either a weekly
(QW) schedule or a schedule of two consecutive daily doses
administered each week (QD.times.2). The group sizes were N=8
mice/group.
[0409] The results are presented in FIG. 7 (data up to day 31) and
FIG. 8. (data up to day 38, i.e., two additional time points
relative to FIG. 7). The data presented in the figures demonstrate
that once weekly IV administration (QW) of liposomal composition
pL-6 comprising carfilzomib (FIG. 7/FIG. 8, liposomal composition
15 mg/kg, QW, open triangles) and QD.times.2 administration of
liposomal composition pL-6 (FIG. 7/FIG. 8, liposomal composition,
10 mg/kg CFZ, QD.times.2, solid circles) induced anti-tumor
responses similar to injectable carfilzomib formulated in SBE-B-CD
(non-liposomal; FIG. 7/FIG. 8, QD.times.2, 5 mg/kg, open squares)
administered on a Day 1/Day 2 schedule (i.e., QD.times.2).
Statistical comparisons between treatment groups and vehicle
controls were made by one-way ANOVA and Bonferroni post-hoc
analysis (significance was p<0.001). The data presented in both
FIG. 7 and FIG. 8 show that the liposomal composition administered
at 15 mg/kg once a week was as efficacious as a liposomal or
non-liposomal composition administered QD.times.2.
[0410] The data in this example demonstrate that liposomal
compositions comprising peptide epoxyketone compounds maintain
efficacy at a reduced dosing frequency relative to a non-liposomal
composition comprising a peptide epoxyketone compound.
Example 12
Induction of Anti-Tumor Response Using Additional Liposomal
Compositions Comprising Entrapped Peptide Epoxyketone
[0411] A. Composition C
[0412] The anti-tumor response of injectable carfilzomib formulated
in SBE-B-CD (non-liposomal) and the liposomal Composition C
comprising liposomes loaded with CFZ was evaluated in mice. Tumors
were established by s.c. injection of RL cells (human non-Hodgkin's
B cell lymphoma cells; passage number <9 and viability >95%
at the time of implantation) in the right flank of BNX mice (n=8
per group). For RL studies, cell suspensions containing
1.times.10.sup.7 cells in a volume of 0.1 mL were injected. Mice
were randomized into treatment groups and dosing initiated when
tumors reached .about.100 mm.sup.3 (RL). Tumors were measured
thrice weekly by recording the longest perpendicular diameters and
tumor volumes were calculated using the equation V (in
mm.sup.3)=(length X width)/2.
[0413] BNX mice bearing established human tumor xenograft derived
from RL cells were treated with either non-liposomal carfilzomib or
liposomal carfilzomib. Drug was administered on either a weekly
(QW) schedule or a schedule of two consecutive daily doses
administered each week (QD.times.2). The group sizes were N=8
mice/group.
[0414] The results are presented in FIG. 9. The data presented in
the figure demonstrate that once weekly IV administration of
liposomal Composition C comprising carfilzomib (FIG. 9, liposomal
composition 15 mg/kg, QW, open circles) and QD.times.2
administrations of liposomal compositions (FIG. 9, liposomal
composition, 5 mg/kg, open diamonds, and 10 mg/kg, QD.times.2, open
triangles) induced anti-tumor responses similar to injectable
carfilzomib formulated in SBE-B-CD (non-liposomal; FIG. 9,
QD.times.2, 5 mg/kg, solid squares) administered on a Day 1/Day 2
schedule (i.e., QD.times.2). Statistical comparisons between
treatment groups and vehicle controls were made by one-way ANOVA
and Bonferroni post-hoc analysis (significance was p<0.001). The
data presented in FIG. 9 show that the liposomal composition
administered at 15 mg/kg once a week (QW) was as efficacious as a
liposomal or non-liposomal composition administered twice weekly
(QD.times.2).
[0415] The data in this example demonstrate that liposomal
compositions comprising peptide epoxyketone compounds maintain
efficacy at a reduced dosing frequency relative to a non-liposomal
composition comprising a peptide epoxyketone compound.
[0416] B. Composition G
[0417] The anti-tumor response of injectable carfilzomib formulated
in SBE-B-CD (non-liposomal) and the pegylated liposomal Composition
G comprising liposomes loaded with CFZ was evaluated in mice.
Tumors were established by s.c. injection of RL cells (human
non-Hodgkin's B cell lymphoma cells; passage number <9 and
viability >95% at the time of implantation) in the right flank
of BNX mice (n=8 per group). For RL studies, cell suspensions
containing 1.times.10.sup.7 cells in a volume of 0.1 mL were
injected. Mice were randomized into treatment groups and dosing
initiated when tumors reached .about.100 mm.sup.3 (RL). Tumors were
measured thrice weekly by recording the longest perpendicular
diameters and tumor volumes were calculated using the equation V
(in mm.sup.3)=(length X width)/2.
[0418] BNX mice bearing established human tumor xenograft derived
from RL cells were treated with either non-liposomal carfilzomib or
pegylated liposomal carfilzomib. Drug was administered on either a
weekly (QW) schedule or a schedule of two consecutive daily doses
administered each week (QD.times.2). The group sizes were N=8
mice/group.
[0419] The results are presented in FIG. 10. The data presented in
the figure demonstrate that once weekly IV administration of
liposomal Composition G comprising carfilzomib (FIG. 10, liposomal
composition 15 mg/kg, QW, solid circles) and QD.times.2
administration of the liposomal composition (FIG. 10, liposomal
composition, 10 mg/kg, QD.times.2, open squares) induced anti-tumor
responses similar to injectable carfilzomib formulated in SBE-B-CD
(non-liposomal; FIG. 10, QD.times.2, 5 mg/kg, open triangles)
administered on a Day 1/Day 2 schedule (i.e., QD.times.2).
Statistical comparisons between treatment groups and vehicle
controls were made by one-way ANOVA and Bonferroni post-hoc
analysis (significance was p<0.001). The data presented in FIG.
10 show that the liposomal composition administered at 15 mg/kg
once a week was as efficacious as a liposomal or non-liposomal
composition administered QD.times.2.
[0420] The data in this example demonstrate that liposomal
compositions comprising peptide epoxyketone compounds maintain
efficacy at a reduced dosing frequency relative to a non-liposomal
composition comprising a peptide epoxyketone compound.
Example 13
Exemplary Criteria for Liposomal Compositions Comprising Peptide
Epoxyketone Compounds
[0421] Exemplary criteria for screening and selection of
advantageous liposomal compositions comprising peptide epoxyketone
compounds include, but are not limited to, the following.
[0422] A. Plasma Half-Life/Pharmacokinetic Data
[0423] Preferred liposomal compositions of the present invention
comprising peptide epoxyketone compounds (e.g., carfilzomib) extend
plasma half-life and provide longer duration of exposure relative
to non-liposomal compositions comprising peptide epoxyketone
compounds (e.g., a non-liposomal composition of carfilzomib
formulated in 10% sulfobutylether beta-cyclodextrin and 10 mM
Citrate, pH 3.5).
[0424] Methods for determination of pharmacokinetic parameters,
including plasma half-life (t.sub.1/2) and area under the plasma
concentration time curve (AUC) are described herein (see, e.g.,
Example 6; Example 9, FIG. 4; Example 11, FIG. 6). Typically,
pharmacokinetic parameters are obtained from rodent studies (e.g.,
using mice or rats). One such screening study to obtain
pharmacokinetic parameters is as follows.
[0425] Plasma t.sub.1/2 of a liposomal composition comprising a
peptide epoxyketone compound is evaluated in 7-8 week old female
BALB/c mice (3-10 mice/time point) following a single i.v.
injection of the liposomal composition comprising the peptide
epoxyketone compound that is being screened. The liposomal
composition is typically administered at one or more drug doses
(e.g., over a dose range of 0.5-50 mg/kg of the peptide epoxyketone
compound). Additionally, a non-liposomal composition comprising the
peptide epoxyketone compound can be included at one or more drug
doses for comparison (e.g., over a dose range of 0.5-15 mg/kg of
the peptide epoxyketone compound).
[0426] The plasma concentration of the peptide epoxyketone compound
is evaluated at a number of time points over a selected time period
(e.g., as shown in FIG. 4). The half-life of the peptide
epoxyketone compound is determined by standard calculations from
this data. AUC is determined by measuring drug clearance rates
using standard calculations.
[0427] B. Pharmacodynamic Profile
[0428] Preferred liposomal compositions of the present invention,
comprising peptide epoxyketone compounds (e.g., carfilzomib)
demonstrate pharmacodynamic profiles comprising (i) at least
equivalent or better biodistribution relative to the non-liposomal
composition comprising the peptide epoxyketone compound (e.g.,
carfilzomib), (ii) at least equivalent or better maximum inhibition
of chymotrypsin-like (CT-L) activity of 20S proteasome relative to
the non-liposomal composition of carfilzomib, and (iii) prolonged
inhibition of the CT-L activity in tissues (e.g., preventing
complete recovery post dose of CT-L activity relative to vehicle,
wherein (a) inhibition of the CT-L activity is observed in one or
more selected target tissues (e.g., adrenal tissue), and (b)
complete recovery from inhibition of CT-L activity is prevented in
one or more selected target tissues (e.g., adrenal tissue) for
greater than 4 hours, preferably at least 6 hours post dosing).
[0429] Methods for determination of pharmacodynamic data, including
biodistribution, maximum CT-L activity inhibition, and prolonged
inhibition in tissues are described herein (see, e.g., Example 5,
FIG. 2A-2D; Example 8, FIG. 3A-3D; Example 11, FIG. 5A-5D).
Typically, pharmacodynamic data are obtained from rodent studies
(e.g., using mice or rats). One such study to obtain
pharmacodynamic data is as follows.
[0430] The pharmacodynamic response of a liposomal composition
comprising a peptide epoxyketone compound is evaluated in 7-8 week
old female BALB/c mice (3-10 mice/time point) following a single
i.v. injection of the liposomal composition (e.g., comprising
carfilzomib) that is being screened. The liposomal composition is
typically administered at one or more drug doses (e.g., over a dose
range of 0.5-50 mg/kg of the peptide epoxyketone compound).
Additionally, a non-liposomal composition comprising the peptide
epoxyketone compound can be included at one or more drug doses for
comparison (e.g., over a dose range of 0.5-15 mg/kg of the peptide
epoxyketone compound).
[0431] The mice are administered selected drug dose(s) of the
liposomal composition. Blood samples and tissues for
pharmacodynamic testing are taken at a number of time points over a
selected time period (e.g., 0.25-168 hour(s); see, e.g., FIG.
5A-5D) after administration of each dose of the liposomal
composition comprising the peptide epoxyketone compound. Typically,
a corresponding control liposomal composition without drug is
included as a control. The pharmacodynamic response is determined
by measurement over time of proteasome activity in blood and tissue
(e.g., whole blood (primarily erythrocytes), adrenal tissue, liver
tissue, heart tissue, and combinations thereof) using a fluorogenic
peptide substrate (LLVY-AMC, as described by Lightcap E S,
McCormack T A, Pien C S, et al., Clin. Chem. 46:673-683 (2000)) to
quantitate the chymotrypsin-like activity of the proteasome. All
samples are normalized relative to the appropriate vehicle (i.e.,
the corresponding composition without carfilzomib). Typically
between three and five tissue samples are evaluated per time point
for each tissue from each mouse for the chymotrypsin-like activity
of the proteasome.
[0432] The biodistribution of the liposomal composition is
determined based on inhibition of the CT-L activity in each
evaluated tissue. The maximum inhibition of CT-L activity is
determined relative to the non-liposomal peptide epoxyketone
compound. Length of time of inhibition and time of complete
recovery of the CT-L activity in tissues (e.g., adrenal tissue) is
evaluated based on the tissue samples over time.
[0433] C. Anti-Tumor Activity
[0434] Preferred liposomal compositions of the present invention,
comprising peptide epoxyketone peptides (e.g., carfilzomib)
demonstrate anti-tumor activity in a human tumor xenograft model
greater than or equal to the non-liposomal composition of the
peptide expoxyketone compound (e.g., carfilzomib).
[0435] Methods for determination of anti-tumor activity are
described herein (see, e.g., Example 11, FIG. 7, FIG. 8; Example
12, FIG. 9, FIG. 10). Typically, anti-tumor data are obtained from
human tumor xenograft rodent studies (e.g., using mice or rats).
One such study to obtain anti-tumor activity data is as
follows.
[0436] Anti-tumor activity of a liposomal composition comprising a
peptide epoxyketone compound is evaluated in mice. The liposomal
composition is typically administered at one or more drug doses
(e.g., over a dose range of 0.5-50 mg/kg of the peptide epoxyketone
compound). Additionally, the non-liposomal composition comprising
the peptide epoxyketone compound can be included at one or more
drug doses for comparison (e.g., over a dose range of 0.5-15 mg/kg
of the peptide epoxyketone compound).
[0437] Tumors are established by s.c. injection of RL cells (human
non-Hodgkin's B cell lymphoma cells; passage number <9 and
viability >95% at the time of implantation) in the right flank
of BNX mice (n=3-10 per group). For RL studies, cell suspensions
containing 1.times.10.sup.7 cells in a volume of 0.1 mL are
injected. Mice are randomized into treatment groups and dosing is
initiated when tumors reach .about.100 mm.sup.3 (RL). Tumors are
measured thrice weekly by recording the longest perpendicular
diameters, and tumor volumes are calculated using the equation V
(in mm.sup.3)=(length X width)/2.
[0438] BNX mice bearing established human tumor xenograft derived
from RL cells are treated with the liposomal composition comprising
the peptide epoxyketone compound. Typically, a non-liposomal
composition comprising the peptide epoxyketone compound is included
at one or more drug doses for comparison (e.g., over a dose range
of 2-15 mg/kg of the peptide epoxyketone compound). Drug is
typically administered on a weekly (QW) schedule and/or a schedule
of two consecutive daily doses administered each week (QD.times.2).
Tumors are measured and tumor volume is determined at a number of
time points (e.g., as shown in FIG. 8) over a selected time period
(e.g., 1-100 day(s)) after administration of the liposomal
composition comprising the peptide epoxyketone compound. Typically,
a corresponding control liposomal composition without drug is
included as a control. The anti-tumor activity for the compositions
is determined by measurement over time of tumor volume.
[0439] Statistical comparisons between treatment groups and vehicle
controls are typically made by one-way ANOVA and Bonferroni
post-hoc analysis.
[0440] As is apparent to one of skill in the art, various
modification and variations of the above embodiments can be made
without departing from the spirit and scope of this invention. Such
modifications and variations are within the scope of this
invention.
* * * * *